Biological control of nematodes

ABSTRACT

The present invention provides a novel isolated and purified fungus  Hirsutella minnesotensis . The present invention also provides a pesticidal composition of an effective amount of an isolated and purified fungal strain of  Hirsutella rhossiliensis  or  Hirsutella minnesotensis  that is capable of controlling nematode infestation and a carrier. Further, the present invention provides methods of controlling nematode infestation.

[0001] This application is a continuation of pending U.S. patentapplication Ser. No. 09/754,097, filed Jan. 4, 2001, entitled,“Biological Control of Nematodes”, which claims the benefit of U.S.Provisional Patent Application Serial No. 60/174,402, filed Jan. 5,2000, entitled, “Biological Control of Nematodes”.

BACKGROUND OF THE INVENTION

[0002] The soybean cyst nematode (SCN), Heterodera glycines, is one ofthe most destructive plant-parasitic nematodes and has been found inmost soybean-growing countries and regions in the world. The northcentral region of the United States is a major soybean-producing regionand the nematode has been reported from all the states except NorthDakota (Noel, 1992; Smolik, 1996). The nematode is a majoryield-limiting factor of soybeans.

[0003] Management of the SCN has largely relied on rotation withnon-host crops and planting resistant cultivars. In many cases, however,rotation and/or use of resistant cultivars are not efficient, or areimpractical. Many factors including biotic and abiotic factors affectthe efficacy of rotation and use of resistant cultivars. Resultsobtained in a recent crop rotation study indicated that 3 years of corndid not appear to be adequate to lower SCN density to a level where anSCN susceptible cultivar could be recommended in Minnesota. Further,increase of years of corn may result in corn yield penalty for thesecond and the following years for unknown reasons.

[0004] Use of resistant cultivars places a selection pressure on thenematode races. Continuous use of resistant cultivars with the sameresistant source may result in race shift and eventually the resistancemay be broken. Furthermore, the nematode can cause a significant yieldloss even to resistant soybean cultivars. Therefore, it is important toreduce nematode density before planting a resistant cultivar.

[0005] Nematophagous fungi have been known for over 100 years and havebeen tested for biological control of plant parasitic nematodes for over60 years (Linford, 1937; Zopf, 1888). Several fungi such as Arthrobotrysspp., Drechmeria coniospora (Drechsler) Gams & Jansson, Hirsutellarhossiliensis Minter & Brady, Paecilomyces lilacinus (Thom.) Samson, andVerticillium chlamydosporium Goddard, have been extensively studied butno successful biological control agents have been developed from thesefungi (Galper et al., 1995; Stirling, 1991).

[0006] Nematophagous fungi have been isolated from various nematodes andlocations. The fungi vary considerably among species and isolates incharacteristics such as virulence to certain nematodes, colonizationability in plant roots, and competitive ability in soil (Boume et al.,1996; Timper and Riggs, 1998). The variability among isolates of H.rhossiliensis, P. lilacinus, and V. chlamydosporium has beendemonstrated (Carneiro and Gomes, 1993; Irving and Kerry, 1987; Tedfordet al., 1994). Waller and Faedo (1993) tested 94 species ofnematode-trapping fungi for their infection of the free-living stage ofanimal-parasitic nematode, Haemonchus contortus Rudolpli in the sheepfecal environment and found only a few species with efficient activity.

[0007]Hirsutella rhossiliensis was first described in 1980 (Minter andBrady, 1980) based on a specimen collected from Wales in 1953. Sturhanand Schneider (1980) reported isolating this fungus from the hop cystnematode, Heterodera humuli Filipjev, and named it Hirsutellaheteroderae (synonym of H. rhossiliensis). The fungus has a wide rangeof hosts including plant-parasitic nematodes, free-living nematodes,entomopathogenic nematodes and mites, although different isolates mayhave different host preferences. Hirsutella rhossiliensis can parasitizea high percentage of nematodes in some locations. This fungus isprobably an obligate parasite in nature and is generally isolated fromonly one species of nematode in a field (Jaffee and Zehr, 1985; Jaffeeet al., 1991; Liu and Chen, 2000a; Sturhan and Schneider, 1980; Timperand Brodie, 1993; Velvis and Kamp, 1995).

[0008]Hirsutella rhossiliensis is a hyphomycetes with simple erectphialides which are swollen at the base and taper towards the apex. Whena host nematode comes into contact with conidia on the phialides, theconidia can attach to the nematode cuticle, and infect the host nematodewithin a few days. Following penetration, the fungus forms an infectionbulb in the nematode cavity, from which assimilative hyphae aredeveloped. After converting nematode body contents to mycelial mass, thefungus may emerge from the nematode cadaver, produce spores, and infectother nematodes. An average of 112 conidia may be formed from myceliumdeveloped from a single juvenile of H. schachtii at 20° C. (Jaffee etal., 1990). KCl increased infection of nematodes by the fungus (Jaffeeand Zehr, 1983). Conidia detached from the phialides may loseinfectivity. Some conidia died shortly after sporulation and others maybe viable and virulent for at least 200 days (Jaffee et al., 1990).Variability of morphology, pathogenicity, and genetics was observedamong isolates (Tedford et al., 1994).

[0009] Parasitism of nematodes by H. rhossiliensis is dependent onnematode density. The percentage of nematodes parasitized by the funguscorrelates positively with host nematode density (Jaffee et al., 1992).The number of conidia attached to cuticle of nematode by H.rhossiliensis correlates with the amount of conidia in the soil. Sincethe fungus is a poor soil competitor, local populations of the fungusmay go extinct unless supplied with some minimum number of nematodes(the host threshold density). Thus, natural epidemics of this fungusamong populations of nematodes develops slowly and only after longperiods of high host densities (Jaffee and Zehr, 1985). Transmission ofspores is greater in loamy sand than in coarse sand (Jaffee et al.,1990). In contrast to the theory that addition of organic matter mayenhance activity of some nematophagous fungi, addition of organic matterto soil decreases parasitism of M. xenoplax by H. rhossiliensis (Jaffeeet al., 1994).

[0010] Many endoparasitic nematophagous fungi produce adhesive spores,which adhere to passing vermiform nematodes, and subsequently infect,and kill the host. Drechmeria coniospora (Drechsler) Gams & Jansson(Drechsler, 1941), Hirsutella rhossiliensis Minter & Brady (Hirsutellaheteroderae Sturhan & Schneider, Sturhan and Schneider 1980; Jaffee andZehr, 1982), and Verticillium banaloides Drechsler (Drechsler, 1941) arewell known species in this group. Only H. rhossiliensis, however,parasitizes high percentages of nematodes in natural soils. The fungusparasitized 80% of Mesocriconema xenoplax Raski in California peachorchard soils (Jaffee et al., 1988) and 90% of Heterodera schachtiiSchmidt J2 in oil-radish fields in Germany (Müller, 1982). Hirsutellarhossiliensis was naturally present in about 25% of the sugarbeet fieldsin Germany, in 17 of 20 fields in a starch-potato-growing area in thenortheastern Netherlands (Velvis and Kamp, 1995), and in 10 of 21sugarbeet fields in California, and may contribute to the suppression ofH. schachtii (Müller, 1984, 1986; Jaffee et al., 1991; Juhl, 1985).Jaffee and Muldoon (1989) also found that penetration of cabbage rootsby H. schachtii was suppressed by 50-77% in loamy sand naturallyinfested with H. rhossiliensis. Hirsutella rhossiliensis has beenisolated from Heterodera humuli Filipjev (Sturhan and Schneider, 1980),H. schachtii (Muller, 1984), Heterodera avenae Woll. (Stirling andKerry, 1983), Heterodera glycines Ichinohe (Chen, 1997), Meloidogynejavanica (Treub) Chitwood (Cayrol et al., 1986), M. xenoplax (Jaffee andZehr, 1982), Rotylenchus robustus (de Man) Filipjev (Jaffee et al.,1991), Xiphinema diversicacaudatum (Micoletzky) Thome (Ciancio et al.,1986), Hoplolaimus galealus Filip. & Schúr. Stek., bacteria-feedingnematodes, soil mites, and soil (Tedford et al., 1994). It has alsoshown to infect Ditylenchus dipsaci (Kuhn) Filipjev, Aphelenchoidesfragariae (Ritz. Bos) Christie, Meloidogyne incognita (Kofoid & White)Chitwood (Cayrol and Frankowski, 1986; Cayrol et al., 1986),Pratylenchus penetrans (Cobb) Filip. & Schur. Stek (Timper and Brodie,1993), Anguina sp. (Cayrol and Combettes, 1983), Anaplectus sp,Cephalobus (Sturhan and Schneider, 1980), and entomopathogenic speciesof Steinernema, Heterorhabditis (Timper et al., 1991) in laboratory andgreenhouse studies.

[0011] The potential of the fungus as biological control agent has beencontroversial. Muller (1982) reported that the fungus might suppresscyst nematodes in some sugar beet fields in Germany. The fungus wasconsidered to be partially responsible for suppression of M. xenoplaxpopulation in some orchards in the southern United States (Zehr, 1985).High numbers and percentages of M xenoplax parasitized by H.rhossiliensis were also found in some California peach orchards (Jaffeeet al., 1989). In greenhouse studies, H. rhossiliensis suppressed G.pallida on potato (Velvis and Kamp, 1996), H. schachtii on cabbage(Jaffee and Muldoon, 1989), and Pratylenchus penetrans Cobb on potato(Timper and Brodie, 1994).

[0012] Results obtained by Tedford et al. (1993), however, indicatedthat long-term interactions between populations of H. rhossiliensis andcyst or root-knot nematodes will not result in biological control. In afield microplot test, H. rhossiliensis failed in suppression of H.schachtii (Jaffee et al., 1996). H. rhossiliensis has been formulated inalginate pellets and used in control of nematodes in laboratory orgreenhouse studies (Lackey et al., 1993; Jaffee et al., 1996). Nocommercial formulation, however, has yet been developed.

[0013] Biological control represents one of the components in anintegrated pest management program and has been shown promise in controlof many other agricultural pests. Especially in view of bans on chemicalnematicides, such as the ban on methyl bromide, there remains acontinuing need for a means to safely and effectively control the spreadof nematodes, specifically of Heterodera glycines. Further, there is along-felt, unresolved need to produce a pesticidal composition that canbe sprayed, or similarly applied, onto crops to control nematodes.

SUMMARY OF THE INVENTION

[0014] The present invention provides a novel isolated and purifiedstrain of fungus Hirsutella minnesotensis. The H. minnesotensis may becapable of controlling plant-parasitic nematodes, such as the nematodeHeterodera glycines, and/or other agricultural pests. The H.minnesotensis may be culture deposit ATCC MYA-31 (CBS 102348) or anyother isolate of the species such as isolates CBS 102457, CBS 102458 orCBS 102459.

[0015] The present invention also provides a pesticidal compositioncomprising an effective amount of an isolated and purified fungal strainof Hirsutella rhossiliensis or Hirsutella minnesotensis capable ofcontrolling nematode infestation, and a carrier. The nematode may be aplant-parasitic nematode, such as Heterodera glycines, but is notlimited to H. glycines. The Hirsutella rhossiliensis may be isolateOWVT-1, specifically culture deposit ATCC PTA-3179 which was depositedin the American Type Culture Collection (ATCC), 10801 University Blvd.,Manassas, Virgina 20110 on Mar. 14, 2001. The H. minnesotensis may beculture deposit ATCC MYA-31 (CBS 102348) which was deposited in theAmerican Type Culture Collection (ATCC), 10801 University Blvd.,Manassas, Virgina 20110 on Mar. 14, 2001, or other isolate. The carriermay be diatomaceous earth, alginate, clay, or other plant and animalproducts as solid formulations. Alternatively the carrier may be aliquid form. An adjuvant or activator may be added into the solid orliquid formulations. Such an adjuvant or activator may increasedispersal, vegetative growth, sporulation, spore germination of thefungus, and/or inhibit soil microbial competitors. The activator may bein a monosaccharide, disaccharide, polysaccharide, amino acid, peptide,peptone, protein, vitamin, other organic compound, or inorganic salt.The Hirsuttella strain may be in the spore and/or mycelium form. Thepesticidal composition may contain an effective amount of at least onestrain of Hirsutella rhossiliensis or Hirsutella minnesotensis that isin the range of about 1×10² to about 1×10¹² spores or colony formingunits (cfu) per milliliter of liquid culture or per gram of solidculture. Alternatively, it may be in the range of about 1×10⁴ to about1×10⁹ spores or cfu per ml or gram, or even in the range of about 1×10⁵to about 1×10⁸ spores or cfu per ml or gram.

[0016] The present invention further provides method for controllingnematodes comprising applying an effective amount of a pesticidalcomposition onto a target plant or onto the situs of a target plant(i.e., the area around the target plant), wherein the pesticidalcomposition comprises an effective amount of an isolated and purifiedfungal strain of Hirsutella rhossiliensis or Hirsutella minnesotensiscapable of controlling nematode infestation and a carrier. Thepesticidal composition is applied at least once, but may be applied aplurality of times in a single growing season, or over the course ofseveral growing seasons.

BRIEF DESCRIPTION OF THE FIGURES

[0017]FIG. 1. Relationship between nematode density in greenhouse potsand parasitism of second-stage Juveniles (J2) of Heterodera glycines byHirsutella rhossiliensis and Hirsutella minnesotensis on agar (A) and insoil in laboratory (B).

DETAILED DESCRIPTION OF THE INVENTION

[0018] Natural enemies of nematodes include fungi, bacteria, viruses andsome microscopic animals such as insects, mites, and nematodes.Nematophagous fungi include trapping fungi that form special devices tocapture and kill nematodes, endoparasites of vermiform nematodes, fungicolonizing eggs and females of sedentary endoparasitic nematodes, andfungi that are antibiotic to nematodes. Most Hirsutella species areparasites of insects and mites. Only H. rhossiliensis and the fungusHirsutella minnesotensis, newly discovered by the present inventors,have been seen to parasitize nematodes.

[0019]Hirsutella minnesotensis Chen et al. is a major parasite of thesecond-stage juveniles (J2) of soybean cyst nematode (SCN), Heteroderaglycines Ichinohe. It has similar characteristics of H. rhossiliensis(Chen et al., 2000; Liu and Chen, 2000a). Hirsutella minnesotensis wasisolated from SCNJ2 collected from a soybean field with a low SCNdensity (Chen et al. 2000).

[0020] Studies were performed to determine distribution, frequencyoccurrence of Hirsutella species in soybean fields in the north centralregion in the United States. Previous reports demonstrated that addinghost nematodes into soil increased detection of the H. rhossiliensis insugar beet fields (Jaffee et al., 1991). For this reason, baitingmethods were used by adding SCN J2 into the soil samples as well asdirect detection methods. Hirsutella minnesotensis was observed in 14%(34 of 237) fields in 27 counties in southern Minnesota. The fungus wasalso detected in South Dakota, Michigan, and Iowa, but not in Missouri,Kansas, and Wisconsin. The fungus appeared to be highly pathogenic toSCN and parasitized a high percentage of J2 in some fields. In a fieldtrial in 1998, H. minnesotensis reduced egg density by 58% at the end ofthe season, which was similar to the efficacy of the nematicide aldicarb(64% reduction); and H. rhossiliensis reduced egg density by 32%. In1999, average yield treated with H. rhossiliensis was 6.2 and 7.0 bu/Ahigher than average of control at two sites; average yield treated withH. minnesotensis was 4.5 bu/A higher than control at one site. In 2000,H. minnesotensis reduced egg density by 39% at the end of the season,and H. rhossiliensis reduced egg density by 20%. The present dataindicate that the fungus is effective as a biocontrol agent.

[0021] Studies were also performed to screen isolates of H.rhossiliensis and H. minnesotensis on agar, in soil, and undergreenhouse conditions for their use as biological control agents againstHeterodera glycines.

[0022] Further, the relationship between J2 density and percentage of J2parasitized by H. rhossiliensis was investigated in plots with a widerange of nematode densities generated with various sequences of soybeancultivars. The J2 density and percentage of J2 parasitized by the funguswere measured. A significant positive relationship between the J2density and percentage of J2 parasitized by the fungus was observed atthe early season and in late season, when there were broad ranges of thenematode densities in the plots. The results also indicate that thefungal population increased when nematode increased on susceptiblecultivars. The increased fungal population, in return, suppressednematode population density. Thus, the fungus was effective inregulating the nematode population.

[0023] Mycopesticide Formulations

[0024] The novel mycopesticide of the present invention can be usedeffectively in diverse formulations, including the agronomicallyacceptable carriers and adjuvants or activators normally employed forfacilitating the activity of ingredients for agriculture applications,recognizing a known fact that the dosage, formulations, mode ofapplication of a chemical agent and other variable may affect itsactivity in any given application. The described mycopesticide can beformulated as a suspension or dispersion, in aqueous or non-aqueousmedia, as a dust, as a wettable powder, as a granule, or as any ofseveral other known types of formulations, depending on the desired modeof application. These pesticide compositions can be applied as sprays,dusts, or granules directly to a plant or its situs where pesticidalactivity is desired.

[0025] The subject fungus, H. minnesotensis or H. rhossiliensis, can beobtained by conventional culture techniques from deposited culturespecimens. To convert it to a form that will facilitate the preparationof the following described compositions, a slurry can be prepared thatcan then be formulated onto a primary agronomically acceptable carrier,e.g., vermiculite, whereby the fungus is incorporated or capsulated ontothe carrier. If desired, the slurry can be used as the concentrate forthe pesticidal composition. The actual concentration of propagules inthe formulated composition is a function of practical consideration suchas the properties of the vehicle or carrier, and the method ofapplication. Certain spore or mycelium concentrations, which aredescribed herein, however, have been found to be preferred. For purposesof formulation and application, an “effective amount” is defined to meanany such quantity of propagules sufficient to infect the target pest andthereby induce the lethal activity described herein.

[0026] The subject material described herein can be applied to a regionto be treated by applying it directly to the soil as pre-emergencetreatment or as post-emergence treatment, or it can be mixed intimatelywith the soil. The preferred mode of treatment is application beforeemergence of the plant foliage. The subject materials described hereincan, for example, be applied to soil in amounts of about 0.1 gallons peracre to 300 gallons per acre, wherein the composition is at aconcentration of about 1×10⁴ to about 1×10¹² spores/cfu per ml as aliquid formulation, or of about 1 lb to about 1,000 lb per acre, whereinthe composition is at a concentration of about 1×10⁴ to about 1×10¹²spores/cfu per gram as a solid formulation.

[0027] As used herein, an “pesticidally effective” amount of the fungalagent is an amount that is sufficient to control nematode infestation.“Controlled” infestation is intended to mean the ability of the fungusaccording to the present invention to control nematode infestation to adegree sufficient to reduce or prevent the ability of nematodes todetrimentally affect the growth of the surrounding plants. However,“controlled”infestation does not necessarily require the completeeradication of all of the nematodes in an area.

[0028] Fungal Agents

[0029] The pesticidal composition of the invention is an effectivepesticidal amount of at least one pathogenic fungal agent capable ofcontrolling nematode infestation, such as Heterodera glycines, combinedwith an agricultural carrier. The fungal agent is a strain of Hirsutellarhossiliensis or Hirsutella minnesotensis. The Hirsutella rhossiliensismay be isolate OWVT-1, specifically culture deposit ATCC No. PTA-3179.The H. minnesotensis may be culture deposit ATCC No. PTA-3180, or otherisolates of the species.

[0030] The method of isolating suitable fungal agents involvesextracting nematodes from soil, plating infected nematodes on agar,transferring fungus growing from a nematode cadaver, and purifying thefungus. The fungus may be maintained on various agar media.

[0031] Suitable Carriers

[0032] The fungal agent is combined with a suitable carrier in aneffective pesticidal concentration. Examples of suitable carriersinclude water, fertilizers, plant-based oils, humectants, orcombinations thereof. Alternatively, the agricultural carrier may be asolid, such as diatomaceous earth, alginate, clay, other plant andanimal products, or combinations, including granules or suspensions.Alternatively, the liquid may be modified to yield a physiologicalsolution. Suitable physiological solutions include sodium phosphate,sodium chloride, sodium acetate, sodium citrate and the like, preferablyin an about 0.001-1 M aqueous phosphate buffer. Other suitablephysiological solutions are well known in the art and would include0.85% sodium chloride. An effective pesticidal amount of the fungalagent is at about 10² to 10¹² spores or cfu per ml or per gram.Alternatively, the concentration is at about 10⁴ to 10⁹, and even atabout 10⁵ to 10⁸ spores or cfu per ml of a liquid medium or per gram ofa solid medium.

[0033] Adjuvants and Germination Activators

[0034] An adjuvant or activator may be added to the pesticidalcomposition of the present invention. The adjuvant or activator providesbetter fungal activities. Preferred adjuvants or activators are thosethat have low phytotoxicity, such as methylated seed oils.

[0035] The adjuvant is dispersed in a liquid suspension containing thefungal agent to yield the present pesticidal composition. The adjuvantis present in an effective plant tissue-penetrating amount that ispreferably within the range of about 0.001% to 10% volume/volume, morepreferably about 0.01% to 6% volume/volume, and most preferably about0.1% to 4% volume/volume of the liquid suspension containing the fungalagent.

[0036] The pesticidal composition may also contain an activator.Examples include monosaccharides, disaccharides, polysaccharides, aminoacids, peptides, peptones, proteins, vitamins, inorganic salts, andother solutes.

[0037] Methods of Forming and Using the Pesticidal Composition

[0038] The pesticidal composition is useful to control nematodes in avariety of environments, especially croplands. The present pesticidalcomposition is formed by combining an effective amount of at least onestrain of a fungal agent with an agricultural carrier, and optionallywith an adjuvant and/or an activator, to form an essentially homogeneousdispersion.

[0039] After the pesticide is formulated, it is applied to thenematode-infested area. The pesticidal mixture may be applied by groundspraying, aerial spraying, painting or brushing, or by hand ormechanical dispersion, including but not limited to backpack or otherhand held devices, hydraulic or air nozzles, granular applicators,electrostatic applicators, controlled droplet applicators (CDA), orultra-low volume (ULV) applicators. The pesticidal composition may alsobe suitable for application by low pressure spraying so that large areasof land may be easily treated.

[0040] The pesticidal mixture is applied in single or repeatedapplications until nematode infestation is effectively inhibited. Theconditions leading to effective nematode inhibition depend, in part, onthe environment. For example, a single application of the pesticidalmixture may be sufficient or a plurality of applications may berequired. The pesticidal composition of the present invention can beapplied to soil, bare ground, plant litter or to plants of any age,including plants that have flowered or senesced. The pesticidal mixtureis applied at a density sufficient to cover the area where nematodegrowth is expected to be observed in amounts from about 0.1 gallons peracre to 300 gallons per acre, wherein the composition is at aconcentration of about 1×10² to about 1×10¹² spores or cfu per ml in aliquid medium, or equivalent in a solid medium. The concentration may bein the range of 10⁴ to 10⁹ spores per ml, or in the range of 10⁵ to 10⁸spores per ml. Nematode growth is effectively inhibited if the majorityof nematodes are infected with the fungal agent and exhibit symptoms ofdisease.

[0041] The following examples are intended to illustrate but not limitthe invention.

EXAMPLE 1

[0042] Distribution and Frequency Occurrence of Hirsutella Species

[0043] A survey was conducted to determine the extent of infestation ofsoybean fields by the fungus Hirsutella in southern Minnesota (Liu andChen, 2000a). A total of 264 soil samples representing 237 fields in 27counties were examined in three experiments. In Test 1, 1,000 J2s wereadded to 50 grams of soil at days 0, 7, 14, and 21, and the nematodeswere recovered at day 28. In Test 2, 2,000 J2s were added to 50 cm³ ofsoil and the nematodes were recovered at day 14. In Test 3, J2s wereextracted from 50 cm³ of soil without addition of nematodes. The J2 wereexamined with an inverted microscope at magnifications of 40-200× forfungal infection. J2 colonized by the fungal mycelium or J2 withattached Hirsutella spores were considered parasitized. Fungi wereisolated from 5 to 15 colonized J2 in each soil for identification tospecies. Two species of Hirsutella, i.e., H. rhossiliensis and H.minnesotensis, were commonly encountered on J2. Combining data from allthree tests, parasitism of J2 by Hirsutella species was observed in soilfrom 46% of fields. Parasitism of J2 by H. rhossiliensis was observed insoil from 43% of fields and by H. minnesotensis in soil from 14% offields. The highest percentage of J2 parasitized by Hirsutella spp. was60%. Addition of J2 into soil increased efficiency of detection of thefungal parasitism.

[0044] Collection of soil samples: About 1,000 soil samples werecollected from soybean fields across southern Minnesota and had beenused for estimating the SCN infestation in the area. Generally, eachsoil sample was taken from soybean root zone with a soil probe or shovelto a depth of 15 to 20 cm in a zigzag pattern in an area of about 5hectares. From these samples, a total of 264 soil samples (representing237 fields in 27 counties) were randomly selected and used for thesurvey of fungal parasites of SCN J2. In a few fields, more than onesoil sample from different areas in the same field were included.

[0045] Test 1: Fifty grams of soil were taken from each of 212 soilsamples, that had been stored in refrigerators (4° C.) for 2 weeks to 6months, and placed into a 240-ml plastic box (70 mm×70 mm×50 mm deep)(Neatway Products Inc., Minneapolis, Minn.). Heterodera glycines wascultured on soybean in autoclaved sandy soil for about 2 months in agrowth room at about 25° C. Newly formed females and cysts were washedwith a vigorously applied water stream through an 800-μm-aperture sieveonto a 250-μm-aperture sieve and extracted by centrifugation in 76%(w/v) sucrose solution. Eggs were released from the cysts by breakingthe cysts in a 40-ml glass tissue grinder (Fisher catalogue No.08-414-10D or equivalent). The eggs were separated from debris bycentrifugation in 35% (w/v) sucrose solution for 5 minutes at 1,500 g.They were treated with a solution containing 100 mg streptomycin, 50 mgof chlortetracycline, and 20 mg of quinolinol per liter of deionizedwater and stored at 4° C. until used for hatching J2. Before hatching,the eggs were rinsed with deionized water and placed in a 4 mM ZnCl₂solution. Within 2 days the hatched J2 were collected and rinsed withsterile deionized water. About 1,000 SCN J2 in 1 ml water were addedinto each box on the surface of soil at days 0, 7, 14, and 21. The boxeswere covered with lids to avoid loss of soil moisture and to preventcontamination. They were maintained at room temperature (22-24° C.)under ambient lights.

[0046] For preparation of soybean root exudates, soybean plants that hadbeen grown in a growth room for 3 weeks were removed from 15-cm-diameterpots and soybean roots were washed with water. About 50 plants wereplaced in a beaker and 300 ml of sterile deionized water was added.After 48 hours, the solution of the root exudates was collected andpassed through a 0.45-μm-pore filter. Fourteen days after first addingthe J2, a 1 ml aliquot of soybean root exudate was added into the soilsurface in each of the boxes to encourage hatching of eggs of naturallyoccurring SCN populations in the soil.

[0047] At day 28, J2 were extracted using a sucrose-flotation andcentrifugation method (Jenkins, 1964). The suspension of nematodes fromeach soil sample was adjusted to 15 ml and stored at 4° C. in arefrigerator until examination. Each nematode suspension wassubsequently placed in one well of 6-well tissue culture plate andexamined at a magnification of 40× to 100× with an inverted microscope.Total J2 and J2 parasitized by H. rhossiliensis and/or H. minnesotensiswere counted. J2 with attached fungal spore(s) or filled with myceliumwere considered parasitized. Parasitism of J2 by the two species wasdistinguished by size and appearance of conidia attached to the nematodecuticle or conidia developing on mycelium growing from a nematodecadaver. To confirm parasitism of J2 by the two species, five to 15 J2with attached fungal spores or filled with mycelium were handpicked fromeach infested soil sample with a needle made from bamboo and placed onpotato dextrose agar (PDA, Difco, Detroit) or corn meal agar (CMA,Difco, Detroit) plates containing 100 mg streptomycin sulfate and 50 mgchlortetracycline per liter of medium for fungal identification.

[0048] In this test, where the J2 were added weekly, H. rhossiliensiswas observed in 77 out of 212 samples (36%), H. minnesotensis wasobserved in 22 samples (10%), and both fungi were observed in 10 samples(5%) (Table 1). The highest percentages of J2 parasitized by H.rhossiliensis and H. minnesotensis in a single soil sample were 50% and35%, respectively. TABLE 1 Frequency occurrence of Hirsutellarhossiliensis (Hr) and/or Hirsutella minnesotensis (Hm) in soilscollected from soybean fields infested or not infested by Heteroderaglycines in each county in southern Minnesota. Total soil Experiment1^(a) Experiment 2^(a) samples^(b) No. of No. of Experiment 3^(a) No. ofTotal fields^(c) No. of samples No. of samples No. of No. of No. ofsamples No. of No. of samples with samples with samples samples sampleswith fields fields with County examined fungi examined fungi examinedwith fungi examined fungi examined fungi Blue Earth 5 1 2 0 1 0 6 1 3 1Brown 22 4 6 1 7 4 24 5 22 5 Carver 2 0 2 0 2 0 Chippewa 1 0 1 0 1 0Cottonwood 3 0 4 0 5 0 5 1 Dodge 2 0 2 0 1 0 4 0 2 0 Faribault 8 4 10 912 1 15 12 15 12 Fillmore 8 2 4 0 8 2 8 2 Freeborn 9 5 4 3 6 1 11 7 11 7Goodhue 1 1 2 0 1 0 2 1 2 1 Jackson 27 12 8 3 12 5 25 11 25 11 Le Sueur5 3 4 0 5 3 2 2 Lincoln 2 0 2 0 2 0 Martin 20 7 11 9 15 5 30 13 23 12Mower 1 1 3 0 1 1 3 1 3 1 Nicollet 4 1 1 0 4 1 3 1 Nobles 2 0 3 0 3 0 10 Olmsted 12 2 3 0 3 0 12 1 12 1 Redwood 3 3 6 1 4 0 7 4 7 4 Rice 1 0 10 1 0 Scott 1 0 1 0 2 0 1 0 Steel 7 6 7 6 9 5 12 9 11 9 Sibley 2 0 4 0 60 6 0 Swift 2 0 1 0 2 0 2 0 Waseca 23 14 12 5 15 2 30 17 28 14 Winona 20 2 0 2 0 Watonwan 38 24 8 6 26 13 40 26 37 25 Total 212 90 98 43 122 37264 114 237 109 % of 42 44 30 43 46 samples with Hr and/or Hm % of 36 4328 40 43 samples with Hr % of 10 10 4 13 14 samples with Hm % of 5 9 2 911 samples with Hr and Hm

[0049] Test 2: Forty-three soil samples that had been used in Test 1 andanother 55 soil samples randomly selected from the 1,000-sample pollwere studied. Fifty cm³ soil from each sample were placed individuallyin 250-ml beakers and 2,000 J2 were added to the soil surface. Thebeakers then were covered with aluminum foil and maintained at roomtemperature (22-25° C.) for 2 weeks. Nematodes were extracted and thepercentage of J2 parasitized by fungi was determined following theprocedures described above.

[0050] In this second test, where 2,000 J2 were added into soil onetime, H. rhossiliensis was observed in 43 out of 98 samples (43%), H.minnesotensis was observed in 10 samples (10%), and both fungi wereobserved in 9 samples (9%) (Table 1). The highest percentage of J2parasitized by H. rhossiliensis in a single soil sample was 60%.

[0051] Test 3: Ninety-six soil samples in which Hirsutella spp. had beenfound in Test and 2 were used in this study. An additional 26 soilsamples randomly selected from the 1,000-sample poll were included. Asubsample of 50 cm³ soil was taken from each soil and J2 were extractedfrom the soil without addition of nematodes. The percentage of J2parasitized by the fungi was determined using the procedures describedabove.

[0052] In this third test, where J2 were extracted directly from thesoil samples, H. rhossiliensis was observed in 34 out of 122 samples(28%), H. minnesotensis was observed in 5 samples (4%), and both fungiwere observed in 2 samples (2%) (Table 1). The highest percentage of J2parasitized by Hirsutella spp. was 36%.

[0053]Hirsutella rhossiliensis was also observed on other nematodes(mainly bacteria-feeding nematodes) in 20 samples, of which 15 samplescontained SCN J2 parasitized by Hirsutella spp. Infection of SCN malesby H. rhossiliensis and H. minnesotensis was also observed.

[0054] Other fungal parasites of J2 were also isolated, includingMonacrosporium drechsleri Tarjan (in nine samples), Hyphochytridiumcatenoides Karling (which has not been reported from nematodespreviously), Verticillium banaloides (Drechsler) Dowsett, Reid & Hopkin,Nematoctonus hamatus Thorn & Barron, and unidentified zoosporic andsterile fungi. These fungi parasitized fewer than 1% of J2.

[0055] Hirsutella species were detected by directly examining the SCN J2that had been attached with fungal spore(s) and/or filled with hyphaerather than by using the method of plating nematode suspensions on agaras described in a previous report (Jaffee et al., 1991). It was assumedthat J2 attached with one or more Hirsutella spores would be eventuallyinfected. This assumption is based on the observation in a previousstudy that a single H. rhossiliensis conidium attached to Ditylenchusdipsaci was sufficient for infection (Cayrol and Frankowski, 1986).Although the possibility could not be excluded that the attached sporewould not infect the SCN J2 and the J2 would be attached by other fungalspores and filled by other fungal hyphae, this possibility was low.First, J2 were filled by distinct hyphae after infection by Hirsutellaspp, but were filled by hyphal fragments, spores, or sporangia afterinfection by zoosporic fungi. Second, in the present study about 500isolates of Hirsutella spp. were obtained, but only a few isolates ofsterile or other fungi were isolated. This indicated that J2 withattached conidia or filled with mycelium were generally parasitized byHirsutella spp. Finally, morphology and size of spores of the twoHirsutella are distinct from each other and no other fungi with similarspores were isolated from the nematode.

[0056] Hirsutella species were detected in 6 of 53 (11%) samples inwhich SCN was not detected, while they were found in 98 of 190 (52%)samples that had been infested by SCN. Since the parasitism of nematodeby H. rhossiliensis is dependent on host density (Jaffee et al., 1992),the fungal population is easier to detect in fields with high nematodedensities than in fields with low nematode densities. In those fieldswhere the fungi were not detected, the fungi may not have beenintroduced or have not established due to the low host nematodepopulation density or absence of the host nematode. It is also possiblethat the fungi existed in a field but were not in the sampled soil dueto limited volume of soil and area sampled. Hirsutella species have beendetected in 17 of 27 counties in Minnesota. Hirsutella species may bepresent in the remaining 10 counties but were not detected due tolimited number of soil samples examined in those counties.

[0057] The baiting methods (Tests 1 and 2) were more effective fordetecting Hirsutella species in SCN J2 than the method of directextraction (Test 3) especially for H. minnesotensis, agreeing with theprevious report on H. schachtii (Jaffee et al., 1991). Similarpercentages of soil samples with infection of Hirsutella spp. weredetected by periodic additions (Test 1) and one-time addition (Test 2)of a large number of H. glycines J2 (Tables 1 and 2). Forty-threesamples were used in both Test 1 and Test 2. Thirty-five out of the 43soil samples had the same results in both experiments. Results of eightsoil samples differed in Experiments 1 and 2, i.e., Hirsutella specieswere found in six samples in Test 1 but not in Test 2, and Hirsutellaspecies were found in two samples in Test 2 but not in Test 1.Hirsutella spp. were found only in 30% of the samples with directextraction in Test 3, although 96 samples used in Test 3 had been foundwith infection of Hirsutella spp. in Experiments 1 and 2. Hirsutellaminnesotensis was detected in 4% of soil samples in Test 3, as comparedwith 10% in Experiments 1 and 2. The highest percentage of parasitizedJ2 in a sample detected by the direct method (36%) was lower than thatdetected by baiting methods (50% and 60% in Experiments 1 and 2,respectively). TABLE 2 Frequency distribution of the parasitism ofHeterodera glycines second-stage juveniles (J2) by Hirsutellarhossiliensis and/or H. minneostensis in soils from southern Minnesota.Experiment 1^(a) Experiment 2^(a) Experiment 3^(a) Total Percentage ofNo. of % of No. of % of No. of % of No. of % of J2 parasitized samplessamples samples samples samples samples samples samples    0 122 57.5 5556.1 86 70.5 263 60.9 0.1-10 52 24.5 30 30.6 30 24.6 112 25.9  11-20- 199.0 8 8.2 2 1.6 29 6.7  21-30 7 3.3 3 3.1 3 2.5 13 3.0  31-40 9 4.2 0 01 0.8 10 2.3  41-50 3 1.4 1 1.0 0 0 4 0.9  51-60 0 0 1 1.0 0 0 1 0.2 >610 0 0 0 0 0 0 0 Total 212 100 98 100 122 100 432 100

[0058] The host range of H. rhossiliensis is relatively broad and mayeven include soil mites (e.g., Sturhan and Schneider, 1980; Tedford etal., 1994). In any given field, however, the fungus is generallyisolated from only one species of host, although many other species ofnematodes are present (Jaffee et al., 1991). Hirsutella rhossiliensisand/or H. minnesotensis were found only in SCN J2 in most soil samplesin the present study, but H. rhossiliensis also was found inbacteria-feeding nematodes in a few samples. The morphology andnutrition requirements of H. rhossiliensis from bacteria-feedingnematodes were somewhat different from that isolated from SCN. There wasno significant difference in morphology between Hirsutella spp. from SCNJ2 and from SCN males. Morphological, pathogenic, and geneticvariability among isolates of H. rhossiliensis has been demonstrated(Tedford et al., 1994). The growth rate of H. rhossiliensis isolatesfrom SCN and bacteria-feeding nematodes were observed to be slower thanthat of the isolate (ATCC 46487) from M. xenoplax.

[0059] In summary, Hirsutella rhossiliensis and H. minnesotensis werethe most common species and either or both species were detected in 17of 27 counties in Minnesota where SCN occurs. Parasitism of SCN J2 byHirsutella species was observed in 43% of the 264 soil samples and 46%of the 237 fields. Hirsutella rhossiliensis was detected in 40% of soilsand 43% of fields and H. minnesotensis was detected in 13% of soilsamples and 14% of fields. Nine percent soil samples and 11% of thefields had both fungi (Table 1). About 13% samples were encountered withover 10% of J2 parasitized by Hirsutella spp. and the highest percentageof J2 parasitized by Hirsutella spp. was 60% (Table 2).

EXAMPLE 2

[0060] Detection of Hirsutella spp. in the North Central Region of theUnited States

[0061] Soil Samples

[0062] A total of 126 soil samples were collected from soybean fields orcorn fields in South Dakota, Iowa, Michigan, Missouri, Wisconsin, andKansas. The soil samples were stored at 4° C. for 2 to 5 months untilused.

[0063] Examination of Soil Samples

[0064] Direct detection: Soil samples were examined directly withoutaddition of bait J2. Native J2 were extracted from a 100-g subsamplewith a sucrose-flotation and centrifugation method. Total J2 and J2parasitized by fungi were counted. Juveniles with attached fungalspore(s) or filled with mycelium were considered parasitized. To confirmparasitism of J2 by a fungus, three to five J2 with attached fungalspore(s) or filled with mycelium were handpicked from each infested soilsample and placed on potato dextrose agar. Fungi growing from the J2were identified.

[0065] Baiting method: In this method, J2 were added into the soil onetime to bait the fungal parasites. One hundred g soil from each samplewere placed individually in a 240-ml plastic box (70×70×50 mm). About2,000 newly hatched SCN J2 in 1 ml water were added to the surface ofsoil in each box. The boxes were covered with lids to reduce loss ofsoil moisture and to minimize contamination. They were maintained atroom temperature (22-24° C.) under ambient light. After 14 days, J2 arebe extracted, and percentage of J2 parasitized by the fungi wasdetermined.

[0066] With the combination of the two detection methods, Hirsutellaspp. colonization of J2 was observed in three soil samples (100%) fromSouth Dakota, seven samples (47%) from Michigan, and nine samples (23%)from Iowa (Table 3). Hirsutella species were not observed in soilsamples from, Missouri, Wisconsin and Kansas. TABLE 3 Frequencyoccurrence of Hirsutella spp. in soils collected from soybean fields innorth central United States. Direct detection^(a) Baiting^(a) Total soilsamples^(b) No. of No. of No. of No. of No. of No. of samples samplessamples samples samples samples State examined with fungi examined withfungi examined with fungi SD 3 3 3 2 3  3 IA 38 0 38 9 38   9^(c) MI 156 15 5 15  7 MO 42 0 42 0 42  0 WI 10 0 9 0 10  0 KA 18 0 17 0 18  0Total 126 9 124 16 126 19 % of samples 7 13 15 with Hr spp.

EXAMPLE 3

[0067] Evaluation of Hirsutella Spp. as a Biocontrol Agent

[0068]Hirsutella rhossiliensis and Hirsutella minnesotensis are twomajor species of endoparasites of Heterodera glycines second-stagejuveniles (J2). The objective of this study was to screen for the mosteffective isolates of the fungi in laboratory and greenhouse forbiocontrol of the nematode. A total of 93 isolates of H. rhossiliensisand 25 isolates of H. minnesotensis were evaluated for parasitism of H.glycines J2 on cornmeal agar. Percentage of SCN J2 parasitized by thefungi varied among the fungal isolates. Most H. rhossiliensis isolatesparasitized a high percentage of J2. The isolates of H. rhossiliensisobtained from bacteria-feeding nematodes, however, generally did notparasitize J2 on agar. Hirsutella minnesotensis parasitized lowerpercentage of J2 on agar than did H. rhossiliensis.

[0069] Forty isolates of H. rhossiliensis and four isolates of H.minnesotensis that parasitized a relatively high percentage of J2 asdetermined on agar were evaluated for their biocontrol potential inlaboratory soil treated with microwave heating, which eliminatesnematodes but does not kill most fungi and bacteria. Most isolatesselected from the agar assay also parasitized a high percentage of J2 inthe soil but there was variation among isolates. Correlation between thepercentage of J2 parasitized on agar and percentage of J2 parasitized insoil was weak, suggesting environmental conditions are important ininfluencing parasitism. Pathogenicity of 14 isolates of H. rhossiliensisand four isolates of H. minnesotensis to the SCN was also investigatedin the greenhouse suing untreated field soil. All isolates of H.rhossiliensis significantly reduced SCN density and increased plantgrowth when compared with non-inoculated soil control. Most isolates ofH. rhossiliensis reduced SCN density and increased plant growth whencompared with 1%-corn-grits control (culture media). One isolate(OWVT-1) of H. rhossiliensis reduced the SCN egg density by 95% and J2density by 98% when compared with 1%-corn-grits control. Isolates of H.minnesotensis, however, neither reduced SCN density nor increased plantgrowth in the greenhouse. The nematode density in the greenhouse potswas negatively correlated with the percentage of J2 parasitized on agar(R²=0.42, P<0.05) and laboratory pasteurized soil (R²=0.47, P<0.05),indicating that the laboratory tests on agar and soil can only partiallyexplain the control efficacy in the greenhouse natural soil. Whilelaboratory tests on agar and in heat-treated soil permitted screening ofpotential isolates from a large number of isolates, the final step ofscreening isolates in the greenhouse using untreated field soil isnecessary to find the most effective isolates as biological controlagents (Liu and Chen, 2000b).

[0070] Fungal isolates and culture. The fungi were isolated from SCN J2in soils collected from fields across southern Minnesota in 1997 and1998 (Liu and Chen, 2000a). A few of the isolates were obtained frombacteria-feeding nematodes. The H. rhossiliensis isolate ATCC 46487originally isolated from adult Criconemella xenoplax (Raski) Luc & Raskifrom soil in South Carolina (Jaffee & Zehr, 1985) was obtained fromAmerican Type Culture Collection. All isolates were maintained on potatodextrose agar (PDA) (Difco, Detroit, Mich.) at 4° C. The fungi werecultured on cornmeal agar (CMA) (Difco, Detroit, Mich.) for agar testsand for preparation of inoculum used in tests in soil in the laboratoryand greenhouse. To prepare inoculum for the soil tests, the fungi werecultured on corn grits (Aunt Jemima, the Quaker Oats Company, Chicago,Ill.) at a ratio of 2:2:1 (sand:corn-grits:water) in 1-liter flasks forabout 2 months at room temperature (22-25° C.). The number of colonyforming units (cfu) per gram of corn grits was determined. Twenty gramsof corn-grits culture per isolate plus 200 ml sterile distilled waterwere blended in a blender (model 57199, Hamilton beach/Proctor-SilexInc.) 3 times, 10 seconds each time. A serial dilution of the fungalsuspension was prepared. One ml of the suspension was placed onto thesurface of a PDA plate. The number of colonies formed on agar werecounted after 7 days.

[0071] Nematode: Soybean cyst nematode race 3 originally from a field inMinnesota was cultured on soybean C.V. Sturdy in sterilized soil in agreenhouse. Newly formed females and cysts were washed with a vigorouslyapplied water stream through an 800-μm-aperture sieve onto a250-μm-aperture and extracted by centrifugation in 76% (w/v) sucrosesolution. Eggs were released from the cysts by breaking the cysts in a40-ml glass tissue grinder (Fisher catalogue No. 08-414-10D orequivalent). The eggs were separated from debris by centrifugation in35% (w/v) sucrose solution for 5 minutes at 1,500 g and then transferredto an antibiotic solution of 100 μg/ml streptomycin sulfate, 50 μg/mlchlortetracycline and 20 μg/ml 8-quinilinol and maintained at 4° C.before being used. Before hatching, the eggs were rinsed with deionizedwater and transferred to a 4 mM ZnCl₂ solution. The nematodes weremaintained at 22-25° C. and the J2 hatched within 2 days were collected.The J2 were rinsed with sterile deionized water and washed into asterile beaker with 4.5 mM KCl before being used in the assays.

[0072] Soil: A field soil was collected from a corn field in Le SueurCounty, Minnesota. Hirsutella rhossiliensis and H. minnesotensis was notobserved on SCN J2 and other nematodes in the soil. Soil for thelaboratory test was treated by microwave heating (1-kg lot of soil in aplastic bag for 1.5 min at 650 W), which eliminated nematodes, butallowed most fungi and bacteria to remain alive. The soil was air-dried,and passed through a 2-mm-aperture sieve. Soil moisture was adjusted to7% (w/w). For the greenhouse test, the field soil was passed through a5-mm-aperture sieve and was used without the heat treatment.

[0073] Test on agar: Ninety-three isolates of H. rhossiliensis (80isolates isolated from SCN J2 and 13 isolates from bacteria-feedingnematodes) and 25 of H. minnesotensis (isolated from SCN J2) were testedfor their ability to parasitize SCN J2 on agar. Discs 0.4-cm diameterwere cut from the actively growing margin of a fungal colony growing onCMA, and one plug per isolate was transferred to the center of each ofthree 10-cm petri dishes containing CMA. The plates were sealed withparafilm and maintained at room temperature. After incubation for 5weeks, approximately 300 SCN J2 in 0.1 ml of 4.5 mM KCl were added tothe fungal culture in each petri dish. The dishes were uncovered for 30minutes in a laminar flow cabinet to allow excess water to evaporate,then covered, and sealed with parafilm. At day 3, J2 were washed offusing 5 ml 0.1% Tween-20 solution. The percentage of J2 colonized by thefungus or J2 with attached fungal spores was determined from 100randomly selected J2 under an inverted microscope.

[0074] Laboratory test in soil: Forty isolates of H. rhossiliensis andfour of H. minnesotensis that parasitized more than 50% J2 on agar(except the isolate FA2-1) were selected for evaluation in laboratorysoil assay. Corn-grits cultures of H. rhossiliensis and H. minnesotensiswere mixed separately with soil at a rate of 1% (corn grits:soil). Adrain hole of 7 mm in diameter was made at the bottom of individual25-ml vials with snap-on caps (Kerr Group Inc., Jackson, Tenn.) and acircle of polyester fabric was placed in the bottom to retain soil(McInnis and Jaffee, 1989). Twenty-five grams of soil were placed ineach plastic vial, and five vials were used for each fungal isolate.After incubation for 3 weeks at room temperature (22-25° C.), 300 newlyhatched J2 in 0.5 ml of 4.5 mM KCl were added onto the surface of thesoil in each tube. After 3 days, the nematodes were recovered from thesoil with a centrifugal-floatation technique (Jenkins, 1964). Thepercentage of J2 colonized by the fungus or J2 with attached fungalspores was determined from 50 randomly selected J2.

[0075] Greenhouse assay: Fourteen isolates of H. rhossiliensis and fourisolates of H. minnesotensis that, except two isolates, parasitized morethan 50% J2 in laboratory agar and soil assays were selected for thisstudy. A corn-grits culture of each fungus was mixed separately withsoil at a rate of 1%. Controls included the soil amended with 1%autoclaved corn-grits and the soil without addition of corn grits orfungal culture. Nematode eggs were added to the soil at a rate of 2,860eggs/100 cm³ soil at the same time. The soil was placed in10-cm-diameter pots (300 ml). Seeds of soybean CV. Sturdy weresurface-disinfected with 0.1% NaOCl₃ for 3 minutes and 2 seeds weresowed in each pot. After 1 week, the soybean plants were thinned toprovide one plant per pot. Density of eggs, density of J2, and thepercentage of J2 colonized by the fungus or with attached fungal sporeswere measured 2 months after inoculation. Plant heights and dry weightswere determined. The nematode densities were expressed as number of eggsand number of J2 per 100 cm³ soil or per gram of plant roots.

[0076] Data analysis: Data of egg and J2 densities were transformed tolog₁₀ (x) values and data of percentage of J2 parasitized by fungi werearcsine (x) transformed before being subjected to analysis of variance(ANOVA). Means were compared with the least significant difference test(LSD) at P=0.05. Contrast analyses were performed to compare theisolates of H. rhossiliensis with H. minnesotensis and compare the fungiwith control. Regression analyses were performed to determine therelationship of fungal parasitism among the tests on agar, in laboratorysoil, and in greenhouse, and to determine the relationship betweenfungal parasitism on water agar or laboratory soil and the nematodedensity in greenhouse pots treated with the fungi.

[0077] Parasitism on agar: Fungal isolates varied substantially in theproportion of SCN J2 infested (Table 4). About 69% of H. rhossiliensisisolates parasitized more than 50% J2 and 18% of isolates parasitizedmore than 80% of J2. The class of parasitism with the highest percentagefrequency (28%) of H. rhossiliensis isolates was 71-80% J2 parasitized(Table 4). In contrast, only 16% of H. minnesotensis isolatesparasitized more than 50% of J2. The class of parasitism with thehighest percentage frequency (48%) of H. minnesotensis isolates was only20-30% J2 parasitized (Table 4). None of the 13 isolates of H.rhossiliensis isolated from bacteria-feeding nematodes parasitized J2within 3 days. TABLE 4 Frequency distribution of isolates of Hirsutellarhossiliensis and Hirsutella minnesotensis among classes of percentageof second- stage juveniles (J2) of Heterodera glycines parasitized onagar and in laboratory soil. on agar in soil H. rhossiliensis H.minnesotensis H. rhossiliensis H. minneasotensis % J2 No. of % No. of %No. of % No. of % parasitized isolates Frequency isolates Frequencyisolates Frequency isolates Frequency 91-100% 0 0 0 0 8 20.0 0 0 81-90%17 18.3 0 0 11 27.5 0 0 71-80% 26 28.0 0 0 7 17.5 2 50 61-70% 12 12.9 28.0 2 5.0 1 25 51-60% 9 9.7 2 8.0 5 12.5 0 0 41-50% 5 5.4 0 0 1 2.5 1 2531-40% 5 5.4 4 16.0 1 2.5 0 0 21-30% 4 4.3 12 48.0 1 2.5 0 0 11-20% 11.1 5 20.0 0 0 0 0  1-10% 1 1.1 0 0 3 7.5 0 0  0-1% 13 14.0 0 0 1 2.5 00 Total 93 25 40 4

[0078] Parasitism in soil: Most fungal isolates that were highlyeffective in the agar assay were also able to parasitize a highproportion (more than 50%) SCN J2 in soil, though there were variationsamong isolates (Table 4). No significant relationship between thepercentage of J2 parasitized on agar and the percentage of J2parasitized in soil was observed (R²=0.07, P=0.09). Among isolates thatwere subsequently used in the greenhouse assay, a positive relationship(R²=0.28, P=0.03) between the percentage of J2 parasitized by the fungion agar and percentage of J2 parasitized in the soil was observed. Someisolates parasitized a high percentage of J2 on agar but showed littleor no ability to parasitize J2 in soil (Tables 4 and 5). Among the H.minnesotensis isolates, percentage of J2 parasitized by one isolate(FA2-1) was significantly lower than that of the other three isolatesboth on agar and in the soil test (Table 5). TABLE 5 Percentage ofsecond-stage juveniles (J2) of Heterodera glycines parasitized byHirsutella rhossiliensis (Hr) and Hirsutella minnesotensis (Hm) in agar,laboratory soil, and greenhouse assays, and the colony-forming units(cfu) of the fungal culture for the laboratory and greenhouse soilsasays. Fungal Laboratory Field soil in cfu species Fungal isolate Agarsoil greenhouse (10⁵) Hr WT24-1 87.8ab 84.0def 13.2fg 67 ATCC 88.7a72.8g 22.8def 49 WT4-1 86.4ab 86.8cde 33.6bcd 71 MA37-1 87.4ab 90.0bcd31.6bcde 61 MA36.4-1 88.3ab 82.0ef 31.6bcde 62 MA30-1 86.0ab 92.0bc27.6bcde 45 ST8-1 84.4abcd 94.8ab 37.6ab 23 WA20-1 85.0abc 35.2h 38.0ab119 LE5.1-1 80.5bcde ND 19.6ef ND WT8-1 83.9abcd 96.8a 23.2cdef 70 FR6-175.8def 87.6cde 32.4bcd 141 MO1-1 74.1ef 83.2def 23.2cdef 44 JA9-177.6cdef 96.0a 28.8bcde 67 OWVT-1 71.7fg 91.6bc 24.4cdef 47 Hm WA23J2-161.3gh 77.2fg 49.2a 22 RW7-1 63.1gh 70.4g  6.8g 64 CRA3-2 51.4h 68.0g35.2bcd 7 FA2-1 17.5I 43.2h 36.0abc 8 Contrast Hr vs. Hm *** *** NS

[0079] Biocontrol efficiency in greenhouse assay: All isolates of H.rhossiliensis tested in field soil significantly reduced the number ofeggs and J2 developed per gram of plants when compared to the soil onlycontrol (Table 6). TABLE 6 Density of Heterodera glycines eggs in potstreated with various isolates of Hirsutella rhossiliensis (Hr) andHirsutella minnesotensis (Hm). Eggs/gram of plant Eggs/100 cm³ of soil %Reduction % Reduction compared compared with controls Fungal withcontrols Corn Soil species Fungal isolate Egg number Corn grits Soilonly Egg number grits only Hr ATCC46487  66,920bcde 11.5 16.4 87,630defg 35.3 88.7 OWVT-1  1,907h 97.5 97.6  3,430j 97.5 99.6 MO1-1 30,063g 60.2 62.4  32,428i 76.1 95.8 FR6-1  28,860fg 61.8 63.9  30,891i77.2 96.0 JA9-1  40,087efg 47.0 49.9  46,783hi 65.5 94.0 LE5.1-1 36,800defg 51.3 54.0  36,301hi 73.2 95.3 WT8-1  33,500efg 55.7 58.2 36,667i 72.9 95.3 MA36.4-1  37,960cdefg 49.8 52.6  50,335fghi 62.8 93.5ST8-1  29,840fg 60.5 62.7  33,318i 75.4 95.7 WA20-1  77,560abcd −2.6 3.1110,493cdef 18.4 85.8 MA30-1  32,880fg 56.5 58.9  42,051hi 69.0 94.6WT4-1  88,240abc −16.7 −10.2  89,275cdefg 34.1 88.5 MA37-1  38,280cdefg49.4 52.2  43,214ghi 68.1 94.4 WT24-1  56,900bcdef 24.7 28.9  81,901efgh39.5 89.5 Hm FA2-1 119,200ab −57.7 −48.9 226,357b −67.1 70.9 CRA3-2112,120ab −48.3 −40.1 199,129bc −47.0 74.4 RW7-1 135,000a −78.6 −68.6285,545b −110.8 63.3 WA23J2-1 112,700ab −49.1 −40.8 175,307bcd −29.477.5 Controls 1% corn grits  75,600abcd 5.6 135,436bcde 82.6 Soil only80,050abcd −5.9 778,250a −474.6 Contrast Hr vs. Control *** *** Hm vs.Control * NS Hr vs. Hm *** ***

[0080] Seventy-one percent (ten of the 14) isolates of H. rhossiliensissignificantly reduced the number of SCN eggs and 43% (six of 14)isolates significantly reduced number of J2 per gram of plants whencompared with the 1%-corn-grits control (Tables 6 and 7). Fifty percent(seven of 14) isolates of H. rhossiliensis reduced the number of eggsper 100 cm³ of soil when compared with the soil only control or1%-corn-grits control. Only twenty-one percent (three of the 14)isolates of H. rhossiliensis significantly reduced the number of J2 per100 cm³ of soil when compared with the soil only control and twoisolates when compared with the 1%-corn-grits control. Egg and J2densities were lowest in pots inoculated with H. rhossiliensis OWVT-1.This fungal isolate reduced the number of eggs per gram of plant by 95%and number of J2 per gram of plant by 98% when compared with the1%-corn-grits control. None of isolates of H. minnesotensis, however,reduced egg and J2 densities in soil or on plants (Tables 6 and 7).TABLE 7 Density of Heterodera glycines second-stage juvenile (J2) inpots treated with various isolates of Hirsutella rhossiliensis (Hr) andHirsutella minnesotensis (Hm). J2/gram of plant % Reduction J2 densityper 100 cm³ of soil compared with % Reduction com- controls FungalFungal pared with controls Soil species isolate J2 number Corn gritsSoil only J2 number Corn grits only Hr ATCC46487  7,532bcde −47.5 −39.3 9,704defgh −5.6 82.1 OWVT-1   268h 94.8 95.1   4811 94.8 99.1 MO1-1 2,700defg 47.1 50.1  2,910ghijk 68.3 94.6 FR6-1  1,478g 71.1 72.7 1,579k 82.8 97.1 JA9-1  4,133efg 19.1 23.6  4,634ijk 49.6 91.4 LE5.1-1 3,106defg 39.2 42.6  3,069hijk 66.6 94.3 WT8-1  3,511defg 31.3 35.1 3,886fghijk 57.7 92.8 MA36.4-1  2,534defg 50.4 53.1  3,353fghij 63.593.8 ST8-1  2,045fg 60.0 62.2  2,278jk 75.2 95.8 WA20-1  6,432abcd −25.9−18.9  8,884cdef 3.3 83.6 MA30-1  2,428efg 52.5 55.1  3,173ghijk 65.594.1 WT4-1  5,768cdef −12.9 −6.7  5,520fghij 39.9 89.8 MA37-1  2,382defg53.4 56.0  2,684hijk 70.8 95.0 WT24-1  4,356cdef 14.7 19.5  6,103efghi33.6 88.7 Hm FA2-1  18764a −267.3 −247.0 36,066ab −292.4 33.5 CRA3-2  7816abc −53.0 −44.5 14,064bcde −53.0 74.1 RW7-1   8480abc −66.0 −56.817,479bcd −90.2 67.8 WA23J2-1  13072ab −155.9 −141.7 20,076abc −118.463.0 Controls 1% corn grits  5,108cdef 5.5  9,192defg 83.0 Soil only 5,408cde −5.9 54,200a −489.6 Contrast Hr vs. Control * *** Hm vs.Control ** NS Hr vs. Hm *** ***

[0081] No parasitism of J2 by Hirsutella species in the soil of eithercontrol was observed. In contrast, in soil inoculated with Hirsutellaspp., between 20% to 50% of J2 were parasitized at the end of the test(Table 7). The percentage of J2 parasitized at the end of the test infield soil in the greenhouse was not correlated with the percentage ofJ2 parasitized on agar or in laboratory soil.

[0082] The number of eggs per gram of plant in the greenhouse pots wasnegatively related to percentage of J2 parasitized on agar (R²=0.42,P=0.0035) and laboratory soil (R=0.47, P=0.0025) (FIG. 1). A similarrelationship was observed between the fungal parasitism of J2 on agar orlaboratory soil and number of eggs per 100 cm³ soil, number of J2 per100 cm³ soil or per gram of plant (data not shown). No significantrelationship between egg density and percentage of J2 parasitized in thepots at the end of the experiment was observed (data not shown).

[0083] Ninety-three percent (13 of 14) H. rhossiliensis isolatessignificantly (P<0.05) increased plant weights and 14% (two of 14)isolates significantly (P<0.05) increased plant heights compared withthe 1%-corn-grits control (Table 8). However, no difference in plantgrowth was observed among plants in H. minnesotensis inoculated soil andthe 1%-corn-grits control. The plant heights and weights were lower(P<0.5) in pots of soil only control than any other treatments includingthe control of 1% corn grits (Table 8). TABLE 8 Growth of soybean plantsin pots infested with Heterodera glycines and treated with variousisolates of Hirsutella rhossiliensis (Hr) and Hirsutella minnesotensis(Hm). Plant height % Increase Plant weight compared with % Increasecompared controls Fungal with controls Corn Species Fungal isolateGrams/pot Corn grits Soil only cm grits Soil only Hr ATCC46487 2.2def38.3 559 68.6efg −6.8 43.5 OWVT-1 1.8fghi 12.3 435 83.0abc 12.8 73.6MO1-1 2.8ab 72.8 724 78.8abcdef 7.1 64.9 FR6-1 2.8ab 75.3 735 81.2abcde10.3 69.9 JA9-1 2.6bcd 59.3 659 90.4a 22.8 89.1 LE5.1-1 3.0a 86.4 78883.8abc 13.9 75.3 WT8-1 2.7abc 66.7 694 87.0ab 18.2 82.0 MA36.4-1 2.3cde40.7 571 74.2bcdefg 0.8 55.2 ST8-1 2.5bcd 55.6 641 76.4bcdefg 3.8 59.8WA20-1 2.2def 37.0 553 82.0abcd 11.4 71.5 MA30-1 2.2defg 35.8 54778.6abcdefg 6.8 64.4 WT4-1 2.9ab 76.5 741 86.4abc 17.4 80.8 MA37-12.6abcd 61.7 671 85.4abc 16.0 78.7 WT24-1 2.2defg 35.8 547 77.0bcdefg4.6 61.1 Hm FA2-1 1.6hi 0 433 69.4defg −5.7 45.2 CRA3-2 1.8ghi 11.3 49382.8abc 12.5 73.2 RW7-1 1.5i −5.0 407 65.4g −11.1 36.8 WA23J2-1 2.0efgh26.3 573 67.0fg −9.0 40.2 Controls 1% corn grits 1.6hi 376 73.6cdefg54.0 Soil only 0.3j −79.0 47.8h −35.1 Contrast Hr vs. Control *** *** Hmvs. *** * Control Hr vs. Hm *** ***

[0084] The density of SCN generally is expressed as number of eggs per100 cm³ of soil. However, reproduction is dependent on available foodsource, i.e., more soybean roots support more nematode reproduction. Inthe present greenhouse test, soybean plants in the soil-only controlgrew poorly and their plant mass was significantly less than in othertreatments. Thus, to minimize bias in the present analysis of nematodepopulation density, the number of eggs or J2 per gram of plant were usedas well as the number of eggs or J2 per 100 cm³ of soil.

[0085] The present results indicate that host specificity exists amongisolates of H. rhossiliensis and H. minnesotensis. Isolates frombacteria-feeding nematodes did not infect SCN J2 within 3 days. Previousstudies demonstrated that H. rhossiliensis and/or H. minnesotensis wereisolated from only one species of host in one field (Jaffee et al.,1991; Liu and Chen, 2000a). Tedford et al. (1994) divided 25 isolates ofH. rhossiliensis into four groups according to their hosts. The isolatesfrom R. robustus and H. galeatus grew slowly in culture, but activelysporulated, produced larger conidia, and were weakly parasitic to H.schachtii, M. javanica, and S. glaseri.

[0086] The methodology applied in the present agar tests and inlaboratory soil might have some limitations that reduced the chances toidentify highly effective biocontrol agents. First, the ratio of sporeson agar or in soil compared to inoculated nematode numbers is too high.In the agar and in soil tests, one nematode probably encountered over1,000 spores. Consequently, the percentage of J2 parasitized may notdifferentiate among isolates. Second, only the percentage of parasitizedJ2 was measured after 3 days. Greater biological and ecologicaldifferences might be detected if more characteristics, such as optimumtemperature, soil moisture, growth rate and sporulation, were measured.Finally, many factors, such as culture media, time and temperature ofincubation, and assay substrate, affect parasitism. For example, Dickieand Bell (1995) examined the effect of nine factors on the outcome ofclassic in vitro screens testing the antagonistic action of endophyticbacterial isolates from grape vines against virulent Agobacterium vitis,and found that nine factors had a significant effect on the diameter ofthe inhibition zones. For these reasons the in vitro screening systemfor nematophagous fungi could be improved.

[0087] The negative relationship between nematode density in soil ingreenhouse pots and percentage of J2 parasitized on agar and inlaboratory soil suggests that the agar and laboratory soil assays canonly partially explain the control efficacy in the greenhouse naturalsoil. While laboratory tests on agar and in soil enable one to screen alarge number of isolates, the step of screening isolates in field soilin the greenhouse is still necessary to find the most effective isolatesas biological control agents.

[0088] The present inventors found that one isolate (OWVT-1) of H.rhossiliensis was highly effective in suppressing SCN density in thenatural soil under greenhouse conditions. The fungus was isolated from asite where soybean has been continuously planted for 27 years and thenematode density was naturally suppressed (Chen, 1997). The long-termmonoculture of soybean may select for fungi having high level ofpathogenicity to the nematode. The present greenhouse assay enabled theinventors to detect the highly pathogenic fungus in natural soil.

EXAMPLE 4

[0089] Determination of Spore Production, Infectivity, SaprophyticActivity, Transmission, and Mortality of H. Minnesotensis.

[0090] Spore production: Three isolates, representing relatively high,moderate, and low pathogenicity to SCN, are used in the present study.Ten agar disks (1-cm diameter) are removed with a cork borer fromplastic petri dishes (10-cm diameter) containing water agar. Onefungus-colonized J2 is placed in a small drop of distilled water on thesurface of plastic in each of the 10 circular areas where agar isremoved. The dish is sealed with parafilm and maintained at 25° C., andeach nematode is examined daily for spore production. Ten replicates (10petri dishes) are used for each isolate.

[0091] Spore infectivity: Detached spores of H. rhossiliensis, a speciessimilar to H. minnesotensis, cannot attach to host nematode and loseinfectivity. This experiment is designed to determine the infectivity ofH. minnesotensis spores detached from phialides. A highly pathogenicisolate is cultured on corn grits. Detached spores are collected. Thefollowing three methods are used: (i) 100 J2 are mixed with 1,000,10,000, and 100,000 spores in 1.5 ml of soil extract solution in amicrofuge tube and centrifuged at 10,000 g for 5, 10, or 20 minutes.(ii) 10,000 spores in 1 ml of sterile soil extract solution are placedon water agar in a petri dish and 100 J2 are added. The plates areincubated at 25° C. for 24 hours. (iii) 100,000 spores are mixed with 25grams of sterile soil and 300 J2 are added on the top of soil. After 2days of incubation at 25° C., the J2 are recovered from the soil. The J2treated with the three methods are examined for spores attached tonematode cuticle.

[0092] Saprophytic growth and spore production in soil: Spore suspensionis prepared. In addition, mycelium suspension without spores isprepared. The fungal colony forming units (cfu) are determined. Soilcollected from a field without SCN and H. minnesotensis is autoclaved,treated with microwave heating (1-kg lot for 1.5 minutes at 650 W tokill microscopic animals but keep fungal and bacterial populations), orused without heating treatment. About 10,000 cfu of fungal suspensionare mixed with 25 grams of soil and placed in vials. At days 0, 3, 7,14, 21, and 28, five vials of each combination of fungal suspension andsoil are assayed for spore production by adding 300 J2 on surface of thesoil in each vial. J2 are recovered from the soil after 3 days andnumber of spores attached to J2 are determined.

[0093] Spore transmission: Soil without SCN and H. minnesotensis iscollected from a field and treated with microwave heating. A highlypathogenic isolate is used in this study. The fungus is cultured on corngrits. The fungal culture is mixed with sterile soil at 1:10, placed in10-cm-diameter petri dishes, and incubated for 1 week before adding SCNJ2 (5,000 J2/dish). After 3 days, the nematodes are recovered andpercent of J2 attached with spores and percent of J2 colonized by thefungus is determined. Fungus-colonized J2 (0, 1, 3, 9, 27, 81, 243, and729 J2/vial) are mixed with 25 grams of soil that is placed in vials.After 1 to 2 weeks, 300 healthy J2 are added on the top of soil in eachvial. The nematodes are recovered from the soil after 3 days andexamined for fungal spore attachment. The probability of sporeacquisition is determined.

[0094] Spore mortality: The design of this experiment is based on theassumption that the fungus cannot grow saprophytically and cannotproduce spores without nematodes as substrate in soil. Thefungus-colonized J2 are prepared and added into vials containing naturalfield soil. Fungus-colonized J2 are added at a level that is needed forabout 90% J2 transmission. At days 10, 20, 30, 60, 90, 120, 150, 210,270, and 330, five vials are assayed to determine spore transmission.

EXAMPLE 5

[0095] Determination of Host Range of H. Minnesotensis.

[0096] Host range is an important character in population dynamics andepidemics of a pathogen and the information is important for developmentof the fungus as a biocontrol agent. Three isolates of H. minnesotensisand one isolate of H. thompsonii var. thompsonii, which is a parasite ofmites and closely related to H. minnesotensis morphologically, is testedon nematodes, insects, and mites.

[0097] A. Nematode hosts: Nematodes used in this experiment includedplant-parasitic nematodes Heterodera glycines, Meloidogyne incognita,Meloidogyne hapla, Meloidogyne javanica, Meloidogyne arenaria,Hoplolaimus sp., and Scutellonema sp.; insect-parasitic nematodesStineriema glaseri and Heterorhabditis bacteriophora; and fungal-feedingnematodes Aphelenchus sp. and Aphelenchoides sp. The fungi were culturedon CMA and the nematodes are exposed to the fungal culture for 3 days.Percentages of nematodes attached by spores or filled with mycelium arerecorded. The J2 were then transferred onto water agar to determinecolonization of J2 by the fungus. All nematodes tested were hosts of thefungus. Percentage of J2 with attached spores or filled with myceliumafter 3 days of exposure to the fungal culture are summarized in Table9. TABLE 9 Percentage of nematodes parasitized by Hirsutellaminnesotensis after 3 days on CMA culture. Nematode FA2-1 MA13-1 WA23-1Aphelenchoides sp. 31.8 54.9 37.3 Aphelenchus sp. 25.2 32.5 43.6Heterodera glycines 11.8 17.7 21.1 Heterorhabditis bacteriophora — — 1.1Hoplolaimus sp. 100 100 — Meloidogyne arenaria 50.0 58.7 58.3Meloidogyne hapla 6.9 36.6 20.8 Meloidogyne incognica 31.3 22.8 29.1Meloidogyne javanica 20.0 21.8 16.7 Scutellonema sp. 11.4 18.6 24.4Stinernema glaseri 3.4 14.0 4.8

[0098] B. Insect hosts: Insect species common in Minnesota, at leastincluding corn root worm, European corn borer and corn ear worm, and oneto two species of soil mites are tested. Because the mode of infectionis unknown if any of the species is a host of H. minnesotensis, threemethods are used for the study.

[0099] (i) Twenty insects and 100 mites of each species are addedseparately onto the fungal culture on CMA, and infection and mortalityare examined daily. The insects and mites on CMA without fungal cultureserve as control.

[0100] (ii) Spore and mycelium suspension in water are prepared fromculture on corn grits. The insects and mites are maintained separatelyon artificial media or on plants, and sprayed with fungal spore andmycelium suspension. Treatment with water is included as control.Infection and mortality are examined daily.

[0101] (iii) The fungus is cultured on corn grits, and mixed withautoclaved soil at 1:10. One hundred cm³ of the mixture is placed in a250-ml beaker and covered with foil. After 1 week, 20 insects and 500mites are introduced separately into the soil in each beaker. Autoclavedsoil mixed with autoclaved fungal culture are included as control. Theinsects and mites are extracted from the soil at days 3, 5, and 7, andtheir infection by the fungus and mortality are examined.

EXAMPLE 6

[0102] Evaluation of Biological Control Efficacy of H. Minnesotensis andH. Rhossiliensis Against SCN in Fields 1998 Field Trial

[0103] A field experiment was conducted at one site in Waseca in 1998.Fungi used in this study included three species of egg-parasitic fungi,Verticillium chlamydosporizim from Florida, ARF 18 from Arkansas, andCylindrocarpon destructans from Minnesota, and two species ofjuvenile-parasitic fungi, Hirsutella rhossiliensis and Hirsutellaminnesotensis from Minnesota. The fungi were cultured on corn-grits,applied in furrow and mixed into soil. Additionally, aldicarb(nematicide) and lorsban (insecticide) were included for a comparisonpurpose. Plots applied with fungal culture medium, without addition ofthe culture medium, and plots with a resistant cultivar Pioneer 9234were included as controls. Each plot consisted of two rows with rowspacing of 30 inches and row length of 15 feet. The plots were separatedby four rows without application of agents. Four replicates were used.Nematode egg density at planting, and egg density and juvenile density 2months after planting and at harvest were determined. Soybean yield wasrecorded. Levels of fungal parasitism of nematode eggs and juvenileswere measured 2 months after planting and at harvest.

[0104] The nematode population response to the fungal and nematicidaltreatment was summarized in Table 10. Resistant cultivar Pioneer 9234was the most effective in reducing the nematode population density (96%reduction). The nematicide aldicarb reduced 64% of eggs and 66% of J2 atthe end of season compared to average of the three controls. There wasno reduction of the nematode density in plots treated with theegg-parasitic fungi, Verticillium chlamydosporium, Cylindrocarpondestructans, and ARF18. However, the J2-parasitic fungi, H.minnesotensis and H. rhossiliensis, significantly reduced the nematodepopulation density. When they were applied alone, H. minnesotensis atdosage of 0.5% reduced 58% of eggs and 56% of J2 at the end of theseason. Hirsutella minnesotensis at dosage of 0.1% was also effective incontrol of the nematode with 45% egg reduction and 62% J2 reduction atthe end of season. Hirsutella rhossiliensis reduced egg density by 32%and J2 density by 31% at the end of the season. When the Hirsutellaspecies applied with the egg-parasitic fungi, the control efficacy wasreduced, suggesting that the egg-parasitic fungi had antagonistic effecton the J2-parasitic fungi. TABLE 10 Population development of thesoybean cyst nematode in field plots treated with nematophagous fungiand nematicides in 1998. Egg population At planting Midseason End seasonEggs/100 cm³ Eggs/100 cm³ % Eggs/100 cm³ % Reproduction rate^(b) AgentDosage soil soil Reduction^(a) soil Reduction^(a) Pf/Pi Pf/Pm Pm/PiVerticillium 0.5% 1,588 2,669 7 18,363 −38 19.1 6.9 3.3 chlamydosporium(V.c.) Cylindrocarpon 0.5% 1,285 3,825 −34 13,450 −1 10.5 3.6 2.9destructans ARF 18 0.5% 1,372 3,994 −40 16,750 −26 13.6 4.5 3.1Hirsutella 0.5% 966 2,206 23 9,125 32 10.2 5.6 2.2 rhossiliensis (H.r.)Hirsutella sp. 0.5% 757 2,256 21 5,566 58 7.7 3.2 2.8 (H.sp.) V.c. 0.1%1,238 3,825 −34 14,763 −11 15.4 4.8 3.3 H.sp. 0.1% 1,285 2,625 8 7,31345 6.4 3.4 2.1 V.c. + H.r. 0.5% + 0.5% 557 2,519 12 10,725 20 24.7 5.14.8 V.c + H.sp. 0.5% + 0.5% 1,066 3,200 −12 13,688 −3 14.7 5.2 3.7 CM60Seed-coating 1,610 4,306 −50 15,450 −16 14.4 3.7 3.9 V.c. + H.r. + CM600.5% + 0.5% 650 2,013 30 13,963 −5 26.2 8.9 3.1 V.c. + H.sp. + 0.5% +0.5% 1,297 3,481 −22 9,050 32 7.7 2.9 3.0 CM60 Aldicarb 1200 g ai/ 7721,200 58 4,788 64 6.5 4.5 1.6 acre Lorsban 1800 g ai/ 785 3,525 −2313,588 −2 19.0 6.0 4.9 acre Resistant cultivar 1,222 838 71 472 96 0.50.6 0.7 Pioneer 9234 Control 1-with 0.5% 1,050 2,719 5 12,500 6 13.9 5.42.6 autoclaved carrier Control 2-with 1% 1,160 1,994 30 14,725 −10 16.79.0 2.2 autoclaved carrier Control 3- 1,013 3,875 −35 12,813 4 15.9 3.94.8 without agent Average of the 1074.2 2862.5 13345.8 15.5 6.1 3.2three controls Juvenile (J2) population Midseason End season J2/100 cm³% J2/100 cm³ % Agent Dosage soil Reduction^(a) soil Reduction^(a)Verticillium 0.5% 254 8 255 13 chlamydosporium (V.c.) Cylindrocarpon0.5% 211 24 262 11 destructans ARF 18 0.5% 268 3 360 −23 Hirsutella 0.5%145 48 202 31 rhossiliensis (H.r.) Hirsutella sp. 0.5% 210 24 128 56(H.sp.) V.c. 0.1% 468 −69 305 −4 H.sp. 0.1% 198 29 112 62 V.c. + H.r.0.5% + 0.5% 250 10 209 29 V.c + H.sp. 0.5% + 0.5% 98 65 176 40 CM60Seed-coating 277 0 215 27 V.c. + H.r. + CM60 0.5% + 0.5% 139 50 228 22V.c. + H.sp. + CM60 0.5% + 0.5% 191 31 208 29 Aldicarb 1200 g ai/acre129 54 98 66 Lorsban 1800 g ai/acre 434 −57 186 37 Resistant cultivar 4584 11 96 Pioneer 9234 Control 1 - with 0.5% 355 318 autoclaved carrierControl 2 - with   1% 173 307 autoclaved carrier Control 3 - without 304254 agent Average of the three 277 293 controls

[0105] Observed percentage of J2 parasitized by the fungi was low. Atmidseason, only in plots treated with H. minnesotensis more then 10% ofJ2 were parasitized. Parasitism of J2 by fungi was generally notobserved at midseason in plots without addition of the Hirsutellaspecies. At the end of season, parasitism of J2 was detected in alltreatments except the plots planted with the resistant cultivar Pioneer9234, in which the J2 density was low (Table 11). TABLE 11 Fungalparasitism of second-stage juveniles (J2) of the soybean cyst nematodein field plots treated with nematophagous fungi and nematicides in 1998.% J2 parasitized End Agent Dosage Midseason season Verticilliumchlamydosporium 0.5% 0 11 (V.c.) Cylindrocarpon destructans 0.5% 2 2 ARF18 0.5% 0 14 Hirsutella rhossiliensis (H.r.) 0.5% 3 12 Hirsutella sp.(H.sp.) 0.5% 10 10 V.c. 0.1% 0 3 H. sp. 0.1% 18 4 V. c. + H.r. 0.5% +0.5% 0 22 V.c. + H.sp. 0.5% + 0.5% 3 14 CM60 Seed-coating 0 4 V.c. +H.r. + CM60 0.5% + 0.5% 3 13 V.c. + H.sp. + CM60 0.5% + 0.5% 4 10Aldicarb 1200 g ai/acre 0 4 Lorsban 1800 g ai/acre 0 16 Resistantcultivar Pioneer 9234 0 0 Control 1 - with autoclaved carrier 0.5% 0 7Control 2 - with autoclaved carrier   1% 0 5 Control 3 - without agent 14

[0106] There was no statistically significant difference of plant growth(stand and soybean yield) among the treatments (Table 12). Emergencerate was the highest in the control plots without addition of agent andthe plots planted with Pioneer 9234. In the plots treated with acombination of V. chlamydosporium, H. rhossiliensis, and the soybeanroot-growth-promoting agent CM60, the emergence rate was only 68%. Thedifference of emergence was probably attributed to the variations ofseeding depth rather the effect of the agents. TABLE 12 Soybean plantgrowth in field plots infested with the soybean cyst nematode andtreated with nematophagous fungi and nematicides in 1998. Plant growthEmer- Yield gence (Bul/ Agent Dosage rate (%) Acre) Verticilliumchlamydosporium (V.c.) 0.5% 88.4 20.7 Cylindrocarpon destructans 0.5%89.1 17.1 ARF 18 0.5% 89.1 23.5 Hirsutella rhossiliensis (H.r.) 0.5%84.1 25.6 Hirsutella sp. (H.sp.) 0.5% 88.2 23.9 V.c. 0.1% 87.9 20.3 H.sp. 0.1% 91.9 19.7 V. c. + H.r. 0.5% + 0.5% 83.6 27.1 V.c. + H.sp.0.5% + 0.5% 88.1 23.0 CM60 Seed-coating 87.7 19.5 V.c. + H.r. + CM600.5% + 0.5% 68.1 24.4 V.c. + H.sp. + CM60 0.5% + 0.5% 80.9 22.6 Aldicarb1200 g ai/acre 88.4 23.0 Lorsban 1800 g ai/acre 80.3 26.0 Resistantcultivar Pioneer 9234 93.1 27.2 Control 1 - with autoclaved carrier 0.5%88.9 22.3 Control 2 - with autoclaved carrier   1% 86.0 19.9 Control 3 -without agent 93.1 24.4 Average of the three controls 89.3 22.2

[0107] 1999 Field Trails

[0108] The methodology applied in 1999 was the same as used in 1998except a few fungal agents were different and fungal parasitism was onlymeasured in the Mid-season (Table 13). TABLE 13 Population density,fungal parasitism of eggs and second-stage juveniles (J2) of Heteroderaglycines, and soybean yield in field plots treated with nematophagousfungi in 1999. Eggs/100 cm³ soil J2/100 % J2 Agent Dosage Pi^(a) Pm^(b)Pf^(c) cm³ soil Parasitized EPI^(c) Yield (bu/a) Steel site Verticillium0.50% 6,313 17,248a^(e) 16,375ab 1,331ab 26.5bcd 4.1a 30.8bcchlamydosporium (Vc) ARF18 0.50% 6,000 13,761abcd  9,975b   856bc 16.0e3.3bc 30.4bcd Hirsutella 0.50% 6,263 15,638abc 28,025a 1,094bc 38.0b3.0c 32.2b rhossiliensis (Hr) Hirsutella 0.10% 6,575 18,956a 20,975a1,356ab 38.0b 3.4abc 32.6b rhossiliensis (Hr) Hirsutella 0.50% 6,52513,819abc 13,025ab 1,238ab 37.5b 4.1a 31.6bc minnesotensis (Hm)Hirsutella 0.10% 6,505 17,360a 15,225ab 1,714a 56.5a 4.1a 30.2bcdeminnesotensis (Hm) Vc + Hm 0.5% + 5,219 15,385ab 14,625ab 1,160ab 36.0bc3.5abc 27.5cde 0.5% Vc + Hr 0.5% + 7,090  8,900cd 14,800ab   998bc19.5de 3.4abc 31.9b 0.5% Temik 1200 g 6,200  7,038d 15,550ab   752c19.5de 3.2bc 30.6bc ai/acre Resistant 5,915  1,425e  1,538c   96d 17.8de4.1a 41.4a cultivar Control without 5,815 12,074abc 15,275ab 1,318ab25.0cde 4.1a 31.3bc agent Control with 0.50% 6,220 16,365ab  9,100b1,162abc 23.0de 3.8abc 26.3de autoclaved corn grits Control with 0.10%5,513 11,413bcd 14,975ab   928bc 17.5de 3.4abc 26.1e autoclaved corngrits Beuveria 0.50% 5,785 17,438a 15,700ab 1,046abc 19.0de 3.8ab 31.1bcbassiana Waseca Verticillium 0.50% 1,383b  1,172b  5,400c   221ab  1.5ef4.1ab 28.3bcde chlamydosporium (Vc) ARF18 0.50% 2,053ab  1,466ab 8,875abc   221ab  2.0def 4.1ab 31.9abcd Hirsutella 0.50% 2,525ab 2,535a 10,150ab   201abc  7.0bc 3.3bc 34.8abc rhossiliensis (Hr)Hirsutella 0.10% 2,024ab  2,169ab  9,300abc   203abc 13.0ab 2.9c 35.4abrhossiliensis (Hr) Hirsutella 0.50% 3,279a  1,808ab  7,000abc   226abc15.5ab 3.4bc 33.3abc minnesotensis (Hm) Hirsutella 0.10% 3,210a  1,194b 5,800bc   290a 18.5a 3.5bc 25.3de minnesotensis (Hm) Vc + Hm 0.5% +1,787ab  1,634ab  6,875abc   145bc  4.1cde 3.6abc 31.1abcd 0.5% Vc + Hr0.5% + 3,218a  2,156ab  7,763bc   231ab  5.5cd 4.1ab 27.6cde 0.5% Temik1200 g 3,403ab  1,994ab 12,100a   218ab  2.5cde 3.5bc 37.5a ai/acreResistant 2,358ab  1,206ab   578d   124c  3.5de 4.5a 31.0abcd cultivarControl without 2,210ab  2,309ab  9,700abc   223ab  3.0cde 3.3bc 24.1deagent Control with 0.50% 3,055a  1,372ab  7,900abc   230ab  1.5def 4.2ab28.5bcde autoclaved corn grits Control with 0.10% 2,757a  1,525ab 7,025bc   227ab  0.0f 3.9abc 27.6cde autoclaved corn grits Beuveria0.50% 1,974ab  1,575ab  9,175abc   259ab  1.5ef 3.7abc 22.2e bassiana

[0109] Overall control efficiency was lower in 1999 than in 1998. InSteele site, egg density 2 months after planting in plots treated with0.5% Verticillium chlamydosporium and 0.5% of H. rhossiliensis was lowerthan corn-grits control (Table 13). No significant reduction of eggdensity by other treatments was observed at Waseca and Steele sites.Parasitism of J2 by Hirsutella was observed in all plots in both sitesregardless the treatments. Percentage of J2 parasitized by Hirsutellaspp., however, was higher in plots treated with H. rhossiliensis or H.minnesotensis than in plots without the fungal treatment. Treatment withegg-parasitic fungi, V. chlamydosporium and ARF18, did not increaseegg-parasitic index at mid-season as compared with controls.Verticillium chlamydosporium inhibited activity of H. rhossiliensis andH. minnesotensis and reduced percentage of J2 parasitized by the fungi.Average yield treated with H. rhossiliensis (0.1% and 0.5%) was 6.2 and7.0 bu/A (27% and 25%) higher (P<0.05) than average yield of control inSteele and Waseca sites, respectively (Table 13). Average yield treatedwith H. minnesotensis was 4.5 (18%) (P<0.05) and 1.3 (4.5%) (notsignificant) but a higher than control at Steele and Waseca sites,respectively (Table 13). Egg-parasitic fungi did not significantlyincrease soybean yield. In contrast, mixture of egg-parasitic fungus V.chlamydosporium with Hirsutella species reduced control efficiency ofHirsutella species, probably due to competition from V. chlamydosporium.

[0110] 2000 Field Trial

[0111] In 2000, one isolated of H. minnesotensis and two isolates of H.rhossiliensis were tested at one site at Waseca. Other proceduresapplied in 2000 were the same as used in 1999. At the end of the season,H. minnesotensis isolate FA2-1 reduced egg density by 39% (significantat P=0.1 but not at P=0.05), and the H. rhossiliensis isolate OWVT-1reduced egg density by 20% (not significant) (Table 14). No yieldincrease in treatments with Hirsutella spp. was observed as comparedwith the control. TABLE 14 Densities of eggs and second-stage juveniles(J2) of Heterodera glycines and percentage of J2 parasitized by fungi,soybean yield in field plots treated with Hirsutella spp., nematicide,and resistant cultivar in 2000.^(a) J2/100 % J2 Eggs/100 cc soil cc soilparasitizd Yield Treatment At planting Midseason At harvest MidseasonMidseason bu/A Hirsutella 2,775a 3,588a 25,800a 257a 5.6b 45.5brhossiliensis (ATCC46487) Hirsutella 1,713a 4,513a  21,675ab 270a 6.4b43.8b rhossiliensis (OWVT-1) Hirsutella 3,097a 4,138a  16,725ab 201a14.2a   44.4b minnesotensis (FA2-1) Aldicarb 2,113a  1,166bc   13,975b 141bc 12.9a   55.5a Resistant 3,188a   303c   879c  38c 4.2b 45.5bsoybean (Freeborn) Corn-grits 1,809a 3,600a 27,225a 161ab 6.5b 44.1bcontrol Non- 3,366a  3,488ab 28,025a 195ab 5.4b 45.2b amendment control

EXAMPLE 7

[0112] Nutritional Requirement of the Nematophagous Fungus HirsutellaRhossiliensis

[0113]Hirsutella rhossiliensis has potential as a biocontrol agentagainst plant-parasitic nematodes and its conidia are necessary forinfection of the nematode. The nutritional requirement for fungalgrowth, sporulation and spore germination remains unknown. Six naturalmedia were examined for growth and sporulation of six isolates, and 20carbohydrates, 18 nitrogen compounds and nine vitamins for growth,sporulation and spore germination of three isolates of H. rhossiliensisin solid and/or liquid cultures. VA, CMA and PDA were the best media forthe fungal growth and MEA, VA and YDA were the best media for thesporulation. Glycogen was the best carbon source for growth and sporegermination of H. rhossiliensis in both liquid and solid cultures andfollowed by sucrose for ATCC46487 and D(+) trehalose for OWVT-1. Inulin,starch soluble, α-cellulose and D(+)trehalose supported good growth forall three isolates. While L-sorbose, D-ribose, citric acid andD-fructose and D(+) galactose reduced the growth of ATCC46487 and OWVT-1and could not be utilized by JA16-1 on agar. D(+)xylose could not beutilized by all of three isolates on agar and in liquid culture. Thebest nitrogen sources were casein and peptone for all isolates of H.rhossiliensis and L-proline for JA16-1 and L-asparagine, L-asparticacid, L-histidine, L-lysine, L-proline, L-phenylalanine and DL-serinefor ATCC46487 in liquid culture. Casein produced the greatest growthrate for the JA16-1 growth on agar. L-asparagine, peptone and L-prolineproduced the greatest growth rate for all isolates on agar. L-cystinedid not support the growth on agar and in liquid culture for allisolates. DL-methionine, L-phenylalanine, DL-methionine, L-lysine,ammonium nitrate, glycine and L-histidine were poor nitrogen sources forthe fungal growth. Vitamins enhanced the growth and sporulation of H.rhossiliensis. The lack of thiamine in the medium with all othervitamins significantly reduced the growth for isolate OWVT-1. Differentisolates of H. rhossiliensis required different carbon, nitrogen sourcesand vitamins for sporulation. The best carbohydrates and nitrogensources for sporulation were D(+)trehalose for isolate ATCC46487,D-sorbitol and DL-threonine for OWVT-1, and D-(+)-cellubiose andL-phenylalanine for JA16-1. Addition of vitamins in media increased thesporulation of H. rhossiliensis. The sporulation was enhanced by lack offolic acid and myo-inositol in the media with all other vitamins forATCC46487, lack of thiamine, folic acid, p-aminobenzoic acid andpyridoxine for OWVT-1 and lack of p-aminobenzoic acid and thiamine forJA16-1 compared to control without any vitamins. The carbohydratesrequired for germination of H. rhossiliensis spores were similar to thatfor growth. Glycogen was the best carbon source for spore germination,D(+)xylose, L-(−)-sorbose inhibited the spore germination of allisolates. Spore germination of H. rhossiliensis was favored by mostnitrogen compounds but was significantly inhibited by L-cystine. Lack ofriboflavin and myo-inositol in media with all other vitaminssignificantly increased spore germination of ATCC46487 and OWVT-1respectively. However, the lack of folic acid, riboflavin, d-biotin andmyo-inositol significantly reduced the spore germination of JA16-1.

[0114] Fungal growth, sporulation, spore germination may vary amongfungi and greatly influenced by media, components of the substrate andculture conditions (Elson et al., 1998; Li & Holdom, 1995; Hayes &Blackburn, 1966; Blackburn & Hayes, 1966). The effects of various carbonand nitrogen sources and vitamins on nematode-trapping fungi have beenstudied (Blackburn & Hayes, 1966; Bricklebank & Cooke, 1969; Coscarelli& Pramer, 1962; Hayes & Blackburn, 1966; Saxena et al., 1989).Arthrobotrys oligospora Friesenis and Arthrobotrys robusta Duddingtonhave simple nutrient requirements, indicating their highly saprophyticability (Blackburn & Hayes, 1966). In contrast, Dactylaria bembicodesDrechsler, a constricting-ring trap-former was unable to utilize eithercellulose or starch and inefficient in utilizing glycogen, maltose orsucrose, indicating its poor saprophytic ability (Gray, 1987;Satchuthananthavale & Cooke, 1967a,b). Little is known about thenutritional requirements of nematode-endoparasitic fungi. Parasitism ofMesocriconema xenoplax Raski by H. rhossiliensis was greatly increasedby KCl solution (Jaffee & Zehr, 1983). The endoparasitic fungiNematoctonus haptocladus Drechsler and Harposporium anguillulae Zopfwere able to grow on simple media containing mineral salts and glucose(Bricklebank & Cooke, 1969).

[0115] The objective of this research was to evaluate the effects ofcommon natural media, various carbon and nitrogen sources, and vitaminson the growth, sporulation, and spore germination of H. rhossiliensis.The natural media are those that containing natural, biologicalproducts. The knowledge of the effects of these nutrients on H.rhossiliensis may help understand population dynamics of the fungus insoil and develop strategies for successful application of the fungus asa biological control agent.

[0116] Fungal isolates: Six isolates of H. rhossiliensis used in thetests on natural media were ATCC46487, OWVT-1, ST3-1, WT21-2, FA2-2, andJA16-1. ATCC46489 was obtained from American Type Culture Collection andoriginally isolated by from Mexocriconema xenoplax collected fromEdgefield, South Carolina. OWVT-1, ST3-1, WT21-2, and FA2-2 wereisolated from H. glycines J2 collected from Waseca, Steele, Watonwan,and Faribault Counties, respectively, and the isolate JA16-1 wasisolated from an unidentified free-living nematode collected fromJackson County during the survey of the fungal parasites of H. glycinesJ2 in Minnesota. The isolates ATCC46487, OWVT-1, and JA16-1 were used intests for their carbon, nitrogen, and vitamin requirements.

[0117] All isolates were preserved on potato dextrose agar (PDA) (Difco,Detroit, Mich.) at 4° C. The fungi were cultured on cornmeal agar (CMA)(Difco, Detroit, Mich.) in 10-cm-diam petri plates before tests onnatural media, and cultured on V-8 juice agar (VA) for the carbon,nitrogen and vitamin tests.

[0118] Basal mineral medium: The medium designed by Blackbure & Hayes(1966) was used as the basal medium for all tests. The medium wascomposed of 10 g maltose, 2.0 g NaNO₃, 0.5 g MgSO₄.7H₂O, 0.5 g KH₂PO₄,0.65 g Na₂HPO₄, and 0.5 g KCl per liter of distilled water for liquidculture, and these compositions with addition of 17 g Bacto agar (Difco,Detroit, Mich.) for agar tests.

[0119] Growth and sporulation of H. rhossiliensis on natural media: Thesix natural media used in this study were PDA, CMA, tryptic soy agar(TSA) (Difco, Detroit, Mich.) malt extract agar (MEA) (Difco, Detroit,Mich.), V-8 juice agar (VA), and yeast dextrose agar (YDA, 1.5% yeastextract, 2% dextrose and 1.7% agar) (MacLeod, 1959), on which growth andsporulation of six isolates of H. rhossiliensis were examined. Circularplugs (0.4-cm-diam.) were removed from colony margins of CMA cultures,and one plug per isolate was transferred to the center of each of threeplates (10-cm-diam.) for each medium. Plates were sealed with parafilmand incubated at room temperature (22-24° C.). Diameters of theresulting colony were measured weekly up to 5 weeks. To determinesporulation, eight plugs (0.4-cm-diam) were removed from each plateafter 5 weeks of incubation and transferred to 10 ml sterile 0.1%Tween-20 surfactant in a 50-ml tube and vigorously agitated on a linearEbe-back shaker (forward and backward shaker) for 15 minutes to dislodgeand suspend the spores. The number of spores per ml was determined withhemacytometer with the aid of microscope at a magnification of400×(Elson et al., 1998).

[0120] Growth of H. rhossiliensis isolates from SCN J2 on VA, CMA andPDA were better than that on YDA, MEA and TSA. Growth of isolates ofATCC46487 from M. xenoplax and JA16-1 from bacterial-feeding nematodewas better on all media except TSA (Table 15). The sporulation of allisolates on MEA, VA and YDA was better than that on PDA and CMA (Table15). TSA was a poor medium for H. rhossiliensis growth and sporulation(Tables 15 and 16). ATCC46487 was the fastest growing isolate on allmedia except TSA, followed by the isolates from SCN J2. JA16-1 was theslowest growing one on VA, CMA and PDA, but was faster than isolatesfrom SCN J2 on YDA and MEA (Table 15). The spore-producing ability ofthe isolates was somewhat different between experiment 1 and 2, butisolate JA16-1 from a bacterial-feeding nematode produced more sporesthan other isolates on all media except TSA in the repeated experiment(Table 15). TABLE 15 Influence of natural media on the colony growth ofH. rhossiliensis (mm/day) Test 1 Test 2 Media OWVT-1 ST3-1 JA16-1 WT21-2ATCC** OWVT-1 JA16-1 WT21-2 FA2-2 VA 0.82^(a) ^(B*) 0.98^(a) ^(A)0.70^(a B) 0.98^(a A) 0.99^(a A) 0.80^(a BC) 0.68^(bc) ^(D) 0.84^(a)^(BC) 0.88^(a AB) CMA 0.71^(b) ^(B) 0.90^(ab) ^(A) 0.68^(a B) 0.90^(a A)1.04^(a A) 0.58^(b B) 0.60^(bc) ^(B) 0.73^(a) ^(B) 0.75^(b B) PDA0.63^(bc) ^(C) 0.80^(b) ^(AB) 0.71^(a BC) 0.86^(a A) 0.85^(a A)0.59^(b D) 0.63^(bc) ^(CD) 0.82^(a) ^(AB) 0.73^(b BC) YGA 0.50^(cd) ^(A)0.52^(c) ^(A) 0.67^(a A) 0.51^(b A) 0.92^(a A) 0.48^(c C) 0.75^(a) ^(B)0.50^(b) ^(C) 0.52^(c C) MEA 0.46^(d) ^(B) 0.50^(c) ^(B) 0.73^(a A)0.49^(b B) 0.95^(a A) 0.40^(c D) 0.73^(bc) ^(B) 0.37^(c) ^(D)0.51^(c CD) TSA 0.25^(e) ^(BC) 0.18^(d) ^(C) 0.36^(b A) 0.30^(c B)0.29^(b AB) 0.19^(d B) 0.34^(d) ^(AB) 0.44^(bc) ^(A) 0.24^(d AB)

[0121] TABLE 16 Influence of the media on the sporulation of H.rhossiliensis (104 spores/cm2) Test 1 Test 2 Media OWVT-1 ST3-1 JA16-1WT21-2 ATCC OWVT-1 JA16-1 FA2-2 MEA 82.3^(a) ^(A) 34.4^(ab) ^(C)30.3^(ab) ^(C) 58.5^(a) ^(B) 23.0^(a) ^(B) 15.2^(ab) ^(B) 58.2^(a A)21.9^(a B) VA 23.6^(b) ^(A) 44.0^(a) ^(A) 38.8^(a) ^(A) 37.1^(b) ^(A)14.4^(b) ^(C) 17.8^(a) ^(BC) 50.3^(a A) 23.7^(a B) YGA 16.4^(bc) ^(B)50.8^(a) ^(A) 25.9^(abc) ^(B) 43.3^(ab) ^(A) 21.9^(a) ^(B) 17.7^(a) ^(B)48.3^(a A) 21.6^(a B) PDA 14.1^(bc) ^(BC) 23.0^(b) ^(AB) 32.3^(ab) ^(A) 9.7^(c) ^(C)  8.1^(bc C) ^(C)  9.2^(bc) ^(BC) 22.7^(b A) 12.9^(b B) CMA 4.1^(c) ^(B) 17.6^(bc) ^(A) 13.1^(bc) ^(A)  6.6^(c) ^(B)  3.1^(cd) ^(B) 3.3^(cd) ^(B) 16.7^(b A)  4.7^(c B) TSA  1.5^(c) ^(A)  1.0^(c) ^(A) 2.1^(c) ^(A)  0.4^(c) ^(A)  0.7^(d) ^(A)  0.0^(d) ^(A)  0.6^(d A) 0.5^(c A)

[0122] Effect of carbon source on growth, sporulation and sporegermination:

[0123] Twenty carbohydrates used in the study were listed in Table 17.An amount of 4 g/liter carbon of each of the test carbon sources wasadded individually into basal medium. NaNO₃ at 0.3296 g/L nitrogen wasused as the nitrogen source in the carbon study. Control was the mediumfree of any carbon. Fifteen ml of medium were added to 50-ml plastictubes for liquid culture and 10-cm-diam petri plates for agar culture.TABLE 17 Effects of carbon sources on growth rate, sporulation and sporegermination of H. rhossiliensis on agar and growth rate in liquidculture after incubation for 5 weeks. On agar Carbon Growth mm-in-diamSporulation 10³/cm² source ATCC46487 OWVT-1 JA16-1 ATCC46487 OWVT-1JA16-1 D-(−)-arabinose  18.0A*    9.7A−  0.0A− 22.3 35.2 27.0a-cellulose 33.7A+ 26.8A+ 19.0A− 1.6 2.1 12.7 D-(+)-cellubiose 33.0A+12.3A   10.0A− 13.8 23.9 88.8 Citric acid 11.3A    9.0A    0.0A− 39.587.5 7.4 D(−)fructose 16.2A    8.5A−  0.0A− 50.2 36.8 0.0 D(+)galactose25.0A+  7.0A−  0.0A− 2.8 7.7 7.4 Glycogen   46.8A++ 34.8A+ 20.5A+ 77.57.9 12.4 D-(+)-glucose 28.3A+  8.0A−  6.5A   7.7 30.7 28.1 Inulin36.7A   25.2A   20.0A   3.5 4.2 10.4 a-lactose 35.8A+  9.8A− 15.8A   2.417.4 37.3 monohydrate D-mannitol 29.5A+ 23.2A   18.0A   15.9 40.6 17.3Maltose monohydrate 34.3A+ 13.7A   11.0A   21.9 34.2 26.6 Melibiose35.0A+ 18.2A   11.8A   41.9 33.7 13.1 D(−) ribose 15.5A    8.0A−  0.0A−61.3 1.4 0.0 D-sorbitol 30.3A+ 11.7A+ 18.3A   64.7 126.0 4.2L-(−)-sorbose  7.8A    6.7A−  0.0A− 62.5 7.4 0.0 Starch soluble  33.0A++ 25.0A+ 19.3A   20.1 6.5 5.0 Sucrose 39.5A+ 21.8A+ 15.0A   38.947.7 6.9 D(+) trehalose    36.5 A++ 30.2A+ 18.3A   172.6 49.2 5.2D(+)xylose  0.0A−  0.0A−  0.0A− 0.0 0.0 2.5 CK (no carbon) 35.0A−26.3A   18.3A− 1.1 3.5 0.0 LSD (P < 0.01) 2.1   2.8   1.4   33.1 38.634.0 On agar Spore germination % In liquid culture Carbon sourceATCC46487 OWVT-1 JA16-1 ATCC46487 OWVT-1 JA16-1 D-(−)-arabinose 48.940.0 32.2 O O O a-cellulose ND ND ND I I O D-(+)-cellubiose 58.9 52.252.2 I I I Citric acid 50.0 52.2 38.9 II II O D(−)fructose 45.6 50.034.4 I I O D(+)galactose 62.2 43.3 43.3 I O O Glycogen 72.2 72.2 77.8 VIV IV D-(+)-glucose 61.1 56.7 65.5 I I O Inulin 54.4 50.0 72.2 II II Ia-lactose 56.7 42.2 68.9 I I I monohydrate D-mannitol 58.9 60.0 61.1 IIIII I Maltose monohydrate 36.7 46.7 64.5 I I I Melibiose 40.0 48.9 61.7III II I D(−) ribose 48.9 45.6 15.5 O O O D-sorbitol 57.8 56.7 67.8 IIIII I L-(−)-sorbose 35.6 26.7 13.3 I I O Starch soluble 47.8 58.9 76.7 IVIII III Sucrose 55.6 52.2 73.4 III II I D(+) trehalose 60.0 62.2 68.9 NDND ND D(+)xylose 21.1 26.7 24.4 O O O CK (no carbon) 55.6 48.9 60.0 I II LSD (P < 0.01) 12.2 13.0 11.7

[0124] Inoculum was prepared by washing spores and hyphal fragments fromV8-juice-agar culture with 0.1% Tween-20 solution and then passedthrough 40-μm-aperture cell strainer (Falcon, Fisher Scientific). Fiftyμl fungal spore and mycelium suspension were used as inoculum for theliquid culture and 10 μl for the agar culture. After incubation for 24hr, 30 spores were randomly observed for germination on the agarculture. Colony forming unites (cfu) originated from the spores and/orhyphal fragments in the suspension were determined. The plates and tubeswere incubated at room temperature for 5 weeks, and then growth wasestimated by visual ratings and colony diameters. The visual ratings forgrowth on agar were: 0=no aerial mycelium; 1=little aerial mycelium; 2=afair amount of aerial mycelium; and 3=abundant arial mycelium. Thevisual ratings for growth in liquid culture were: 0=no visible growth;1=a little growth with separated hyphae, but no colony formed; 2=smallseparated colonies or hyphal mass formed; 3=hyphae grew or coloniesjoined together to form pellets, and total volume of pellets was lessthan 2.5 ml; 4=pellet diameters were larger than 1 cm, and total volumeof the pellets was more than 5 ml; 5=hyphal mass was more than 5 ml. Thecolony diameter was measured after 5 weeks for the agar culture, andthen 1 ml of 0.1% Tween-20 solution was added onto each colony to washthe spores into a 1.5-ml centrifuge tube. Spores were counted by usinghemacytometer with the aid of microscope, and the number of spores persquare centimeter was determined (Elson et al., 1998). Growth of fungion some media was not measurable, but spores were produced in the areaof inoculation (approximately 0.6 cm in diam). The sporulation persquare centimeter was calculated from the inoculation area in thissituation.

[0125] Glycogen was the best carbon source for growth of H.rhossiliensis among 20 carbon compounds tested in both liquid and solidcultures and followed by sucrose for ATCC46487 and D(+) trehalose forOWVT-1. Inulin, starch soluble, α-cellulose, and D(+)trehalose supportedgood growth for all three isolates. The good growth was supported byα-lactose monohydrate, melibiose, maltose monohydrate andD-(+)-cellubiose for ATCC46487, D-mannitol, sucrose and melibiose forOWVT-1 and D-sorbitol, and D-mannitol, α-lactose monohydrate, andsucrose for JA16-1. While L-sorbose, D-ribose, citric acid andD-fructose and D(+) galactose were poor carbon source for ATCC46487 andOWVT-1 and could not be utilized by JA16-1 on agar. D(+)xylose could notbe utilized by all isolates on agar and in liquid culture, andD-(−)-arabinose and D(−) ribose could not be utilized in liquid culture.Growth of H. rhossiliensis on control without any addition ofcarbohydrates was fastest on the solid media, but the resultant colonywas quite sparse (Table 17).

[0126] Effect of nitrogen source on growth, sporulation and sporegermination: Eighteen nitrogen compounds were used in his study (Table18). Sucrose at 4 g/L carbon was used as the carbon source for thenitrogen study. Experimental procedures were the same as that employedin the carbon study, except that the amount of each nitrogen source wascalculated so as to provide nitrogen of 0.3296 g/L. Controls were themedium without nitrogen and the medium without both nitrogen and carbon.TABLE 18 Effects of the nitrogen sources on growth rate, sporulation andspore germination of H. rhossiliensis on agar and growth rate in liquidculture after incubation for 5 weeks. On agar Nitrogen Growth mm-in-diamSporulation 10³/cm² source ATCC46487 OWVT-1 JA16-1 ATCC46487 OWVT-1JA16-1 NaNO₃  39.2A+* 21.7A 15.3A− 10.0 35.9 6.4 L-Arginine 42.8A+ 19.5A15.8A   3.5 19.1 1.4 L-Asparagine 51.8A+ 20.8A 16.5A   4.5 18.7 8.3L-Aspartic acid 43.8A+ 16.8A 13.3A+ 5.2 3.6 10.9 Casein    NDA++ 23.0A  NDA ND 43.6 ND L-cystine 0.0    0.0  0.0   12.3 0.0 3.9 Glycine  41.5A++   10.7A+  7.5A   10.2 33.9 1.3 DL-Glutamic acid 40.8A+ 18.7A12.8A+ 8.7 2.6 29.8 L-Histidine   36.3A++   12.8A+  8.5A− 1.8 2.1 3.4L-Lysine 31.9A+    9.3A−   9.8A− 1.7 0.0 2.1 DL-Methionine 11.7A    0.0 0.0   3.2 0.0 10.8 Peptone    46.2 A++ 16.8A 24.8A+ 6.2 13.9 15.5L-proline 47.2A+   23.8A− 17.0A   7.3 9.6 30.9 L-Phenylalanine 34.3A+  17.5A+ 11.7A   7.9 15.9 79.0 DL-Serine   45.3A++ 17.2A 10.2A   7.9 3.12.4 DL-Threonine   25.4A++    9.0A−   8.3A− 13.8 136.5 26.4 L-tyrosine35.7A+ 19.3A 15.7A   15.4 22.4 31.2 Urea 43.5A+ 16.7A 14.0A+ 5.2 10.60.4 Ammonium nitrate   35.7A++   17.2A+ 11.0A− 10.3 4.6 2.3 CK(no N)29.8A+   25.3A+   13.7A++ 0.5 22.3 41.7 CK(no N & C) 42.8A−   24.7A−17.5A− 5.6 3.7 13.3 LSD (P < 0.01) 5.7    3.2  2.1   9.5 25.8 39.7 Onagar Spore germination % In liquit culture Nitrogen source ATCC46487OWVT-1 JA16-1 ATCC46487 OWVT-1 JA16-1 NaNO₃ 86.7 55.6 73.3 IV II IL-Arginine 91.1 76.7 86.6 V III IV L-Asparagine 93.3 70.0 85.5 VI III IVL-Aspartic acid 93.3 53.3 90.0 VI III IV Casein 96.7 44.4 90.0 VI V VL-cystine 14.4 5.6 4.4 O O 0 Glycine 75.6 60.0 93.3 VI III IIIDL-Glutamic acid 74.4 48.9 90.0 VI III V L-Histidine 82.2 65.6 82.2 VIII II L-Lysine 86.7 50.0 90.0 IV III III DL-Methionine 74.4 63.3 70.0III I I Peptone 96.7 66.7 92.2 VI V V L-proline 86.7 60.0 86.7 VI III VL-Phenylalanine 77.8 68.3 86.7 VI IV O DL-Serine 84.4 72.2 87.8 VI IVIII DL-Threonine 93.3 48.9 83.3 V III IV L-tyrosine 91.1 67.8 86.7 V IIIII Urea 91.1 64.4 85.6 IV II II Ammonium nitrate 93.3 57.8 91.1 V IIIIII CK(no N) 73.3 71.1 87.8 I I I CK(no N & C) 85.6 45.6 81.1 O O O LSD(P < 0.01) 8.9 12.9 8.3

[0127] The nitrogen compounds resulting in the faster growth in bothliquid and agar cultures were casein and peptone for all isolates of H.rhossiliensis, and L-proline for JA16-1 and L-asparagine, L-asparticacid, L-histidine, L-lysine, L-proline, L-phenylalanine and DL-serinefor ATCC46487 in liquid culture. Peptone was the best for the JA16-1growth on agar. L-asparagine and L-proline were the best nitrogensources for all isolates on agar. Casein supported good growth, althoughthe data for ATCC46487 and JA16-1 were not included because the inoculumdrop on agar spread out. L-cystine could not be utilized by all isolateson agar and in liquid culture. DL-methionine could not be utilized byOWVT-1 and JA16-1 and inhibited ATCC46487 growth on agar.L-phenylalanine could not be utilized by JA16-1 in liquid culture.DL-methionine and L-lysine reduced the growth of all isolates. Ammoniumnitrate inhibited ATCC46487 growth and glycine and L-histidine inhibitedthe growth of OWVT-1 and JA16-1 (Table 18).

[0128] Sporulation was affected by different nitrogen sources for thethree isolates. The best nitrogen compounds for sponilation wereDL-threonine for OWVT-1 and L-phenylalanine for JA16-1. L-tyrosine,DL-threonine supported better sporulation of ATCC46487 but notsignificantly different from control without nitrogen (Table 18). Sporegermination of all isolates was well supported by most nitrogencompounds but were significantly inhibited by L-cystine (Table 18).

[0129] Effect of vitamin on growth, sporulation and spore germination:Nine vitamins were used in this study (Table 19). Vitamin requirementwas determined by excluding one vitamin at each time from the basalmedium plus all test vitamins. All of the vitamins selected were addedto the basal medium at 200 μg/L except that biotin and myo-inositol wereadded at 5 μg/L and 5 mg/L, respectively. Thiamin and 4-aminobenzoicacid were sterilized by filtering with a 0.45-μ-aperture filter (Fisher,Scientific) and added to the sterile medium. Other vitamins were addedinto the medium prior to heat sterilization. Controls were the mediumwith all vitamins and the medium without any vitamins (Saxena et al.,1989). Sucrose at 4 g/L carbon and NaNO₃ at 0.3296 g/L nitrogen wereused as the carbon and nitrogen sources, respectively, in the vitaminstudy. Other procedures of the experiment were the same as used in thecarbon study. TABLE 19 Effects of the vitamins on growth rate,sporulation and spore germination of H. rhossiliensis on agar and growthrate in liquid culture after incubation for 5 weeks. On agar VitaminGrowth rate mm-in-diam Sporulation 10³/cm² excluded ATCC46487 OWVT-1JA16-1 ATCC46487 OWVT-1 JA16-1 p-Amino-benzoic  41.5A++* 28.3A+ 16.7A+29.9 14.2 35.1 acid (PABA) d-Biotin (V.H) 39.7A++ 27.7A+ 16.3A+ 29.511.7 14.1 Folic acid 39.3A++ 28.7A+ 19.7A+ 59.7 15.4 24.2 myo-inositol40.8A++ 31.5A+ 18.3A+ 41.9 8.3 16.7 Nicotinic acid 40.5A++ 27.7A+ 20.0A+30.1 10.7 12.2 DL-pantothenic 39.5A++ 29.3A+ 18.8A+ 28.6 9.9 17.1 acidPyridoxine (VB6) 40.5A++ 29.5A+ 17.2A+ 31.1 13.0 16.5 Riboflavin, UPS(VB2) 41.0A++ 30.2A+ 20.2A+ 30.6 11.1 13.2 Thiamine (VB1) 38.5A++ 24.7A+20.3A+ 30.1 15.7 30.4 CK (all Vitamins) 39.3A++ 28.7A+ 18.8A+ 28.2 9.922.6 CK (no Vitamins) 38.5A++ 21.8A   15.5A+ 16.3 6.2 4.4 LSD (P < 0.01)5.2    2.7   5.6   19.4 6.4 22.2 On agar Vitamin Spore germination % Inliquit culture excluded ATCC46487 OWVT-1 JA16-1 ATCC46487 OWVT-1 JA16-1p-Amino-benzoic 82.2 51.1 84.4  II** II I acid (PABA) d-Biotin (V.H)80.0 50.0 90.0 II II I Folic acid 82.2 61.1 87.8 II II I myo-inositol72.2 64.4 84.4 II II I Nicotinic acid 77.8 57.8 94.4 II II IDL-pantothenic 72.2 55.6 88.9 II II I acid Pyridoxine (VB6) 74.4 50.090.0 III II I Riboflavin, UPS (VB2) 91.1 60.0 85.6 II I I Thiamine (VB1)75.6 58.9 91.1 II II II CK (all Vitamins) 77.8 55.6 93.3 II II II CK (noVitamins) 71.1 43.3 88.9 II II I LSD (P < 0.01) 8.4 7.4 4.4

[0130] All vitamins promoted OWVT-1 growth significantly. Lack ofthiamine in the medium with all other vitamins significantly reduced thegrowth in OWVT-1. The vitamins seemed increase the growth of ATCC46487and JA16-1 but were not statistically significant (Table 19).

[0131] Addition of vitamins in the media generally increased thesporulation of all isolates but was not statistically significant. Thesporulation was enhanced by lack of folic acid and myo-inositol forATCC46487. Lack of thiamine, folic acid, p-aminobenzoic acid andpyridoxine increased sporulation of OWVT-1 and lack of p-aminobenzoicacid and thiamine increased sporulation of JA16-1 (Table 19).

[0132] The lack of riboflavin and myo-inositol in the media with allother vitamins significantly increased spore germination of ATCC46487and OWVT-1 respectively. However, the lack of folic acid, riboflavin,d-biotin and myo-inositol significantly reduced spore germination ofJA16-1 (Table 19).

[0133] All publications, patents and patent documents are incorporatedby reference herein, as though individually incorporated by reference.The invention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the scope of the invention.

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What is claimed is:
 1. A biologically pure culture of Hirsutellaminnesotensis.
 2. The culture of claim 1, wherein the H. minnesotensisis capable of controlling nematode infestation.
 3. The culture of claim2, wherein the nematode is a plant-parasitic nematode.
 4. The culture ofclaim 3, wherein the nematode is Heterodera glycines.
 5. A pesticidalcomposition comprising an effective amount of a biologically pureculture of a fungal strain, wherein the fungal strain is Hirsutellarhossiliensis ATCC PTA-3179 or Hirsutella minnesotensis capable ofcontrolling nematode infestation and a carrier.
 6. The pesticidalcomposition of claim 5, wherein the nematode is a plant-parasiticnematode.
 7. The pesticidal composition of claim 6, wherein the nematodeis Heterodera glycines.
 8. The pesticidal composition of claim 5,wherein the fungal strain is Hirsutella rhossiliensis ATCC PTA-3179. 9.The pesticidal composition of claim 5, wherein the fungal strain isHirsutella minnesotensis.
 10. The pesticidal composition of claim 5,wherein the carrier comprises diatomaceous earth, alginate, or clay. 11.The pesticidal composition of claim 5, wherein the carrier is a liquidor solid carrier.
 12. The pesticidal composition of claim 5, furthercomprising at least one adjuvant or activator.
 13. The pesticidalcomposition of claim 12, wherein at least one adjuvant is watermiscibleor water-dispersable.
 14. The pesticidal composition of claim 5, whereinthe Hirsutella rhossiliensis ATCC PTA-3179 or Hirsutella minnesotensisis in the spore form, mycelium form, or a mixture of both.
 15. Thepesticidal composition of claim 14, wherein the effective amount ofHirsutella rhossiliensis ATCC PTA-3179 or Hirsutella minnesotensis is inthe range of about 1×10² to about 1×10² spores or colony forming units(cfu) per ml or per gram of the pesticidal composition.
 16. Thepesticidal composition of claim 14, wherein the effective amount ofHirsutella rhossiliensis ATCC PTA-3179 or Hirsutella minnesotensis is inthe range of about 1×10⁴ to about 1×10⁹ spores or cfu per ml or per gramof the pesticidal composition.
 17. The pesticidal composition of claim14, wherein the effective amount of Hirsutella rhossiliensis ATCCPTA-3179 or Hirsutella minnesotensis is in the range of about 1×10⁵ toabout 1×10⁸ spores cfu per ml or per gram of the pesticidal composition.18. The pesticidal composition of claim 5, further comprising agermination, growth, or infection activator.
 19. The pesticidalcomposition of claim 18, wherein the activator is a monosaccharide,disaccharide, polysaccharide, amino acid, peptide, peptone, protein,vitamin, other organic compound, or inorganic salt.
 20. A method forcontrolling nematodes comprising applying an effective amount of apesticidal composition into soil before planting, or onto a target plantor onto the situs of a target plant, wherein the pesticidal compositioncomprises an effective amount of a biologically pure culture of a fungalstrain, wherein the fungal strain is Hirsutella rhossiliensis ATCCPTA-3179 or Hirsutella minnesotensis capable of controlling nematodeinfestation and a carrier.
 21. The method of claim 20, wherein thenematode is a plant-parasitic nematode.
 22. The method of claim 20,wherein the nematode is Heterodera glycines.
 23. The method of claim 20,wherein the fungal strain is Hirsutella rhossiliensis ATCC PTA-3179. 24.The method of claim 20, wherein the fungal strain is Hirsutellaminnesotensis.
 25. The method of claim 20, wherein the carrier comprisesdiatomaceous earth, alginate, or clay.
 26. The method of claim 20,wherein the carrier is a liquid or solid carrier.
 27. The method ofclaim 20, further comprising at least one adjuvant or activator.
 28. Themethod of claim 27, wherein at least one adjuvant is water-miscible orwater-dispersable.
 29. The method of claim 20, wherein the Hirsutellarhossiliensis ATCC PTA-3179 or Hirsutella minnesotensis is in the sporeor mycelium form, or both.
 30. The method of claim 29, wherein theeffective amount of Hirsutella rhossiliensis ATCC PTA-3179 or Hirsutellaminnesotensis is in the range of about 1×10² to about 1×10¹² spores orcfu per ml or gram of the pesticidal composition.
 31. The method ofclaim 29, wherein the effective amount of Hirsutella rhossiliensis ATCCPTA-3179 or Hirsutella minnesotensis is in the range of about 1×10⁴ toabout 1×10⁹ spores or cfu per ml or gram of the pesticidal composition.32. The method of claim 29, wherein the effective amount of Hirsutellarhossiliensis ATCC PTA-3179 or Hirsutella minnesotensis is in the rangeof about 1×10⁵ to about 1×10⁸ spores or cfu per ml or gram of thepesticidal composition.
 33. The method of claim 20, further comprising agermination activator, wherein the germination activator affects fungalactivities.
 34. The method of claim 33, wherein the germinationactivator is a monosaccharide, disaccharide, polysaccharide, amino acid,peptide, peptone, protein, vitamin, other organic compound, or inorganicsalt.
 35. The method of claim 20, wherein the pesticidal composition isapplied at least once.
 36. The method of claim 20 wherein the nematodesare controlled for multiple growing seasons.
 37. A biologically pureculture of Hirsutella rhossiliensis ATCC PTA-3179.